Methods and reagents for improvement of detecting amyloid beta-peptides

FIELD: medicine.

SUBSTANCE: group of inventions relate to medicine and deals with method of diagnosing neurodegenerative disease in individual, including the following stages (i) determination of one or several parameters, selected from group, consisting of 3ab40 or value of calculated parameter, selected from group, consisting of 2ab40+3ab40, 2ab40+3ab40+2ab42+3ab42 and 1ab40+2ab40+1ab42+2ab42; (ii) comparison of parameter value with standard value, corresponding to value of said parameter in standard sample; and (iii) diagnostics of neurodegenerative disease, in case if increase of parameter value in comparison with standard value is observed. Group of inventions also deals with method of detecting stage, preceding neurodegenerative disease, method of differentiating neurodegenerative disease from stage, preceding said neurodegenerative disease.

EFFECT: group of inventions provide high sensitivity and specificity of detection methods.

13 cl, 12 ex, 14 dwg, 12 tbl

 

The scope to which the invention relates

The present invention relates to the field of immunoassays, and more particularly to methods of increasing the sensitivity of the immunoassay conducted to identify amyloid beta peptides in biological fluids.

Prior art

Alzheimer's disease (AD) is a progressive generative disease of the Central nervous system characterized by progressive and progressive loss of memory followed by disruption of the functioning of the motor functions of the limbs and of the whole organism and, ultimately, fatal. This disease, obviously, is the most common cause of dementia, which occurs in 1-6% of the population aged 65 years and above and in 10-20% of the population aged over 80 years.

BA is different from other types of dementia by several pathological features, including progressive formation of senile plaques in the extracellular space between neurons in the brain of patients. In the core of plaques formed by amyloid deposits formed mainly of peptide fibrils, consisting of 40-42 amino acids called amyloid β-peptide (includes AB), which is surrounded by degenerated neurite and glial cells. Such a peptide clicks�ized as a result of proteolytic processing of the precursor protein, called protein precursor of β-amyloid (βΑΡΡ).

BA can be classified according to age manifestation of the disease, i.e., at a younger age (under 60 years) and older age (over age 60 years) with autosomal dominant inheritance, that is, as hereditary or sporadic BA. Early manifesting a hereditary form BA can be associated with a known mutation in the genes encoding βΑΡΡ, presenilin 1 and presenilin 2 (and located, respectively, on chromosomes 21, 14 and 1). These classifications are not mutually exclusive. The most frequent forms are the sporadic form, which is manifested at a later age.

In clinical practice, the diagnosis of BA put on clinical criteria, based on the presence of typical clinical signs and exclusion of other types of dementia using the methods of neuroimaging and blood tests. The accuracy of the diagnosis made on the basis of these criteria, is acceptable, although in accordance with research carried out after the autopsy the brain was found that 10-20% of patients diagnosed with BA suffered and other disease. In addition, modern methods of diagnostics can be executed only in the case when the neurodegenerative process is �is running, the patient suffers multiple forms of dementia, and brain damage is so extensive that it limits the choice of therapeutic measures. For definitive diagnosis requires post-mortem study of brain tissue.

Taking into account the fact that these AB accumulates in the brain of ad patients and is a Central element in the pathogenesis of BA, it should be noted that this protein is considered as the most suitable candidate for its use as a biomarker for ad. However, the use of these AB as a biomarker of BA present in plasma, is associated with a problem, namely that the concentration of these AB peptides (includes AB(1-40) and includes AB(1-42)) in serum are extremely low, and therefore there are no tests that have sensitivity sufficient to perform a reliable detection of the indicated peptide molecules.

To determine the levels of amyloid beta peptides in biological samples was conducted a variety of analyses (see, for example, the methods described Scheuner et al. (Nature Med., 1996, 2:864-870); Tamaoka A, et al. (J. Neurol. Sci., 1996, 141, 65-68); Suzuki, N. et al. (Science, 1994, 264: 1336-1340); WO200722015, Vanderstichele H, et al. (Amyloid, 2000, 7, 245-258); Fukomoto y col. (Arch. Neurol. 2003, 60, 958-964); Mehta et al. (Arch. Neurol. 57, 2000, 100-105); Mayeux, R. et al. (Ann Neurol. 1999, 46, 412-416); Lanz, T. A. and Schacthter, J. B. (J. Neuroscience Methods, 2006, 157:71-81), and in WO200750359, WO0162801,WO0315617, WO0246237, WO0413172. However, all ELISA assays, known in the present time, have a low limit of detection, in all probability, not entering the unambiguous interval of values PG/ml, which is sufficient for the detection of Αβ40 and Αβ42 in CSF, as well as for the detection of these molecules in the plasma of patients suffering from hereditary BA, and are unsuitable for the detection of Αβ42 in the plasma of patients suffering from sporadic BA, in which the concentration of Αβ42 in the plasma is much lower.

To date, analyses performed, only one peptide includes AB are below the detection limit (definite values in the range of PG/ml, as described in WO200646644 and WO2009015696.

In WO200646644 described electrochemiluminescent (goes) the "sandwich"analysis, where the mAb 21F12 (recognizes amino acids 33-42 Αβ42) associated with magnetic fields, which are then used to capture the peptide Αβ42 in a sample containing Αβ42, and is subjected to contacting with mAb 3D6 associated with the ruthenium complex. Then the number of bound antibodies 3D6 detect by fluorescent radiation emitted by the ruthenium complex in the communication of electrical energy. Applying this analysis allows the authors of the present invention to detect at least 0.5 PG/ml standard Αβ42. However, if the same analysis applied to compare Αβ42 � plasma samples from patients with asthma and in healthy patients then any significant differences between patients of these two groups could not be observed, resulting in the authors of the present invention came to the conclusion that the number of intact Αβ42 in serum is very low, due to its degradation, and returned to competitive ELISA analysis using mAb 21F12, which gives a lower level of sensitivity in the range of values of the order of ng/ml.

In WO2009015696 described a highly sensitive ELISA-sandwich-analysis, which detected antibody are contacted with a Biotin-labeled reagent specific to a given antibody. This reagent are contacted with the streptavidin linked to horseradish peroxidase. Then detects the peroxidase activity colorimetric method using TMB reagent or fluorescent method using QuantaBlue.

In WO2006053251 described method for the determination of molecules of amyloid beta-peptide in the sample, where the method comprises contacting the sample with a denaturing agent, extraction of the peptide pool of the mixture "sample-denaturing agent, separation of the molecules of amyloid beta-peptide from the specified pool and the determination of the number of molecules of amyloid beta-peptide. This method requires a phase separation of peptides prior to their definition, which increases the time of implementation of this method � costs.

It is therefore necessary to develop an improved immunological tests and kits for the detection of peptides derived from these AB that will solve the problems associated with the use of known methods and kits, and in particular to develop assays and kits with a sensitivity sufficient for reliable detection of these AB-peptides in the plasma of patients suffering from sporadic BA.

Description of the invention

In its first aspect, the present invention relates to a method for diagnosing a neurodegenerative disease in an individual, the detection of a condition prior to a neurodegenerative disease, or differentiation of neurodegenerative diseases from a state that is prior to the specified neurodegenerative disease, where the method comprises the steps:

(i) determining one or more parameters selected from the group consisting of:

(a) the level of one or more free amyloid beta-peptides in a biological sample of the individual;

(b) levels of aggregates of one or more free amyloid peptides in a biological sample of the individual and the specified one or more amyloid beta peptides associated with macromolecular components present in the specified biologist�'s weekend sample where the reported levels of aggregates is determined by evaluating the number of said one or more amyloid beta peptides in cell-free fractions of the specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the amyloid beta peptide or amyloid beta-peptides of the components present in the biological sample,

(c) the level of one or more amyloid beta peptides associated with cells in a biological sample of the individual, where the specified level is determined by isolating a cellular fraction of the specified biological sample, contacting the specified cell to the specified fraction of the sample with a protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the amyloid beta peptide or amyloid beta-peptides from cells present in a given sample;

(ii) comparing a value of at least one of the parameters (b) or (c), or the value of the parameter calculated by an arithmetic combination of at least two parameters (a) to(c) with a reference value corresponding to the value of the specified parameters (b) or (c) the calculated or specified parameter in the reference model; and

(iii) diagnosis of neurodegenerative diseases; detection condition�I, prior neurodegenerative disease; or differentiation of neurodegenerative diseases from a state that is prior to the specified neurodegenerative disease, if there is a change in the parameter value or values of the calculated parameter is compared with a reference value.

In its second aspect, the present invention relates to a kit for detection of amyloid beta peptides in the biological sample, where the specified set includes:

(i) protein-solubilizers agent and

(ii) at least an antibody against amyloid beta-peptide.

Brief description of graphic material

Figure 1: A-F. Built by the points on the curves of measurements for (A) UP Αβ1-40, (B) DP Αβ1-40, (C) CBAβ1-40, (D) UP Αβ1-42, (E) DP Αβ1-42 and (F) CB Αβ1-42, obtained in two independent laboratories (Lab1 and Lab2). The majority of points is close to the line of concordance, indicating a significant and almost the exact degree of compliance measurements. On each chart in the box below on the right is listed the correlation coefficient and the concordance (CCC) and 95% confidence intervals.

Figure 2: Direct markers of Αβ1-40 (A), Αβ1-42 (B) and calculated markers Αβ1-40 and Αβ1-42 (C).A. Concentration (PG/ml) of free Αβ1-40 in serum (FP), the levels of total Αβ1-40 in plasma (including free Αβ1-40 and Αβ1-40, associated with components of the plasma) (TP) and Αβ1-40, related to�Adami (CB) in healthy control individuals (HC), in patients suffering from mild disruption of cognitive abilities (MCI), and patients with Alzheimer's disease (AD).V.Concentration (PG/ml) of free Αβ1-42 in serum (FP), General levels of Αβ1-42 in the plasma (including free Αβ1-42 and Αβ1-42, associated with components of the plasma) (TP) and Αβ1-42, associated with the cells (CB) in healthy control individuals (HC), patients suffering from mild disruption of cognitive abilities (MCI), and patients with Alzheimer's disease (AD).C. Values, PG/ml, for an aggregate total Αβ1-40 in plasma (obtained by contacting the plasma sample with a protein-solubilizers agent) plus associated with cells of Αβ1-40 (TP+CB Αβ1-40), total Αβ1-42 in the plasma (obtained by contacting the plasma sample with a protein-solubilizers agent) plus associated with cells of Αβ1-42 (TP+CB Αβ1-42), or the amount of the aggregated TP+CB Αβ1-40 and Αβ1-42 (TP+CB Αβ1-42). H, M and A denote significant values (p<0,05) for healthy control (LC) of individuals, for patients with a weak violation of cognitive abilities (MCI) and ad patients, respectively. * indicates p<0.01.

Figure 3: A-F. Built by the points on the curves of quantities (A) DP Αβ1-40, (B) CB Αβ1-40, (C) UP Αβ1-42, (D) DP Αβ1-42, (E) T40 and (F) Τ-βΑΡΒ for ZK-individuals, patients with MCI and patients with asthma. Figures with * indicate the values obtained for individual patients in groups of MCI and BA, �have a quiet, for accuracy of performance, not given in the same scale on the y-axis. The horizontal line represents the boundary values obtained for patients with MCI and ZK-individuals.

Figure 4: ROC curve for the marker lab40 in patients with MCI/HC. The area under the ROC curve = 0,510.

Figure 5: ROC curve for the marker 2ab40 in patients with MCI/HC. The area under the ROC curve = 0,778.

Figure 6: ROC curve for the marker 3ab40 in patients with MCI/HC. The area under the ROC curve = 0,458.

Figure 7: ROC curve for the marker lab42 in patients with MCI/HC. The area under the ROC curve = 0,576.

Figure 8: ROC curve for the marker 2ab42 in patients with MCI/HC. The area under the ROC curve = 0,667.

Figure 9: ROC curve for the marker 3ab42 in patients with MCI/HC. The area under the ROC curve = 0,744.

Figure 10: ROC curve for the marker 3ab42 in patients with MCI/HC. The area under the ROC curve = 0,508.

Figure 11: ROC curve for markers 2ab40+3ab40 in patients with MCI/HC. The area under the ROC curve = 0,830.

Figure 12: ROC curve for markers 2ab42+3ab42 in patients with MCI/HC. The area under the ROC curve = 0,713.

Figure 13: ROC curve for markers 2ab42+3ab42 in patients with MCI/HC. The area under the ROC curve = 0,777.

Figure 14: ROC curve for markers 2ab40+3ab40+2ab42+3ab42 in patients with MCI/HC. The area under the ROC curve = 0,848.

Detailed description of the invention

The authors present invention it was unexpectedly found that the dilution of the plasma sample buffer leads to an increase in detectable levels of Αβ1-40 and Αβ1-42. N� limited to any theory, it can be noted that such a dilution of the plasma leads to a change in ionic strength and molecular interactions with the sample with the subsequent release of Αβ1-40 and Αβ1-42, associated with proteins and other components of plasma.

Thus, the increase in the values measured after dilution of the plasma may be due to the detection of these AB-peptides released from proteins and other components of plasma, and can be interpreted as the magnitude of the overall level of these AB in plasma.

In any case, these results show that the levels of these AB-peptide in the blood is much higher than the levels that could be obtained by a simple analysis in undiluted plasma. Full definition of General levels βΑΡΒ in the blood should include quantitative assessment of free peptides in plasma, peptides associated with plasma proteins, and peptides related to blood cells. This comprehensive quantitative assessment of the various components βΑΡΒ will more accurately measure these AB levels in the blood and gives the opportunity to better understand the complex regulatory mechanism of these AB-peptides in healthy and sick person. In addition, the levels of these pools of amyloid beta-peptides, as well as some of the calculated parameters obtained by arithmetic summation of the concentrations of the different pools and concentrations of free amyloid beta-peptides, can �be used to determine whether the patient suffers from a neurodegenerative disease or condition prior to a neurodegenerative disease, and to differentiate neurodegenerative disease from a state that is prior to the specified neurodegenerative disease.

Diagnostic method according to the invention

Thus, in its first aspect, the present invention relates to a method for diagnosing a neurodegenerative disease in an individual, the detection of a condition prior to a neurodegenerative disease, or differentiation of neurodegenerative diseases from a state that is prior to the specified neurodegenerative disease, where the method comprises the steps:

(i) determining one or more parameters selected from the group consisting of:

(a) the level of one or more free amyloid beta-peptides in a biological sample of the individual;

(b) levels of aggregates of one or more free amyloid peptides in a biological sample of the individual and the specified one or more amyloid beta peptides associated with macromolecular components present in the biological sample, where the reported levels of aggregates is determined by evaluating the number of said about�nogo or more amyloid beta peptides in cell-free fractions of the specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for stimulation of dissociation of the amyloid beta peptide or amyloid beta-peptides of the components present in the biological sample;

(c) the level of one or more amyloid beta peptides associated with cells in a biological sample of the individual, where the specified level is determined by isolating a cellular fraction of the specified biological sample, contacting the specified cell to the specified fraction of the sample with a protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the amyloid beta peptide or amyloid beta-peptides from cells present in a given sample; and

(ii) comparing a value of at least one of the parameters (b) or (c), or the value of the parameter calculated by an arithmetic combination of at least two parameters (a) to(c), with a reference value corresponding to the value of the specified parameters (b) or (c), or the calculated parameter in the reference model; and

(iii) diagnosis of neurodegenerative diseases, the detection of a condition prior to a neurodegenerative disease, or differentiation of neurodegenerative diseases from a state that is prior to the specified neurodegenerative disease, if there is a change in the value of the parameter or value�HN calculated parameter is compared with a reference value.

Used here, the term "diagnosis" includes evaluating the predisposition of an individual to the development of disease, determining the presence of disease in an individual, but also the prediction of outcome of disease in an individual suffering from this disease. As is well known to specialists in this field, the establishment of such a diagnosis in an individual usually can not be 100% accurate, although, preferably, it was accurate. However this term means that a statistically significant group of individuals can be identified as individuals suffering from this disease or having a predisposition to the disease. If this group is statistically significant, then it can be easily determined by the expert with the use of several well known methods of statistical evaluation, for example by determining confidence intervals and p-values, application of t-student test, Mann-Whitney, etc., a Detailed description is given in the publications Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The quantity p is preferably equal to 0.2, and 0.1 or 0.05.

As used herein, the term "individual" refers to all of the belly�th, classified as mammals, including, but not limited to, domestic and farm animals, primates and humans, such as humans, primates, except humans, cows, horses, pigs, sheep, goats, dogs, cats or rodents. The preferred individual is a man, i.e., man or woman of any age or race.

As used herein, the term "neurodegenerative disease" means a condition or disorder in which there is a loss of neural cells resulting in their death, which leads to deterioration of cognitive function or lesion or dysfunction of the nervous system, or to complications that can be characterized as neurological, neurodegenerative, physiological, mental or behavioural disorders. Suitable neurodegenerative diseases that can be diagnosed by methods according to the invention, include, but are not limited to, age-related macular degeneration; the disease of familial multiple sclerosis-Jakob disease; Alzheimer's disease; dementia caused by radiation therapy; axonal damage; acute disseminated depression caused by dysfunction of the cerebral cortex, the alpha synucleinopathies, cerebral ischemia, Huntington's disease, permanent focal ischemia of the cerebral cortex, the regeneration peripheries�their nerves, model postoperational status, spinal cord injury, sporadic amyotrophic lateral sclerosis, transmissible spongiform encephalopathy.

In a preferred embodiment of the invention, the neurodegenerative disease is Alzheimer's disease. The term "Alzheimer's disease" (or "senile dementia" means mental illness, associated with a specific degenerative disease of the brain, which is characterized by the presence of senile plaques, Narineh tangles and progressive loss of neurons and is clinically manifested by progressive memory loss, confusion, behavioral disorders, incapacity, gradual physical deterioration and ultimately leads to death. Patients suffering from Alzheimer's disease, were identified using criteria NINCDS-ADRDA (on a scale CDR = 1, on a scale MMSE between 16 and 24 points and atrophy of medial temporal plot area (as defined by MRI) >3 points on a scale of Selena).

As used herein, the term "stage prior to a neurodegenerative disease" means a condition that can be defined as a transient condition that is observed in a normal individual, the state, defined as a neurodegenerative disorder, and that x�racterized the presence of certain signs and symptoms of neurodegenerative disorders or subgroups of signs and symptoms, observed in patients suffering from a neurodegenerative disorder. In a preferred embodiment of the invention, a stage that is earlier than the specified neurodegenerative disease, is a weak violation of cognitive abilities (hereinafter referred MCI), which means the transfer stage from cognitive disabilities in natural aging to early Alzheimer's disease. Patients generally identify as patients suffering from MCI if they meet the clinical criterion Mayo (CDR = 0.5, which indicates atrophy of the medial temporal plot area (determined by MRI), and this rating is 3 points higher on a scale of Selena that indicates a pattern of hypometabolism in the parietal and/or temporal region identified using positron emission tomography, carried out with 18-Tordesillas (PET-FDG)) (presumably, BA).

The term "differentiation of neurodegenerative disease from a stage prior to a neurodegenerative disease" refers to the ability to distinguish the patient with the state prior to a neurodegenerative disease or condition in the prodromal period of the disease, from patients already suffering from this disease. In the specific case where the neurodegenerative disease is a disease �of lzgamer, the method according to the invention allows to differentiate Alzheimer's disease from the prodromal stage of the disease, known as weak violation of cognitive abilities (MCI).

In its first stage, the method according to the invention includes determining at least one parameter selected from the group consisting of:

(a) the level of one or more free amyloid beta-peptides in a biological sample of the individual;

(b) levels of aggregates of one or more free amyloid peptides in a biological sample of the individual and the specified one or more amyloid beta peptides associated with macromolecular components present in the specified biological sample, where the reported levels of aggregates is determined by evaluating the number of said one or more amyloid beta peptides in cell-free fractions of the specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the amyloid beta peptide or amyloid beta-peptides of the components present in the biological sample;

(c) the level of one or more amyloid beta peptides associated with cells in a biological sample of the individual, where the decree�nny level is determined by isolating a cellular fraction of the specified biological sample, contacting the specified cell to the specified fraction of the sample with a protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the amyloid beta peptide or amyloid beta-peptides from cells present in a given sample.

As used herein, the term "amyloid beta peptide" is synonymous with "A-beta", "Abeta", "beta AP", "A-beta peptide," or "includes AB-peptide" means a family of peptides, which are the main chemical constituents of senile plaques and vascular amyloid deposits (amyloid angiopathy) present in the brain of patients with Alzheimer's disease (AD), patients with down syndrome and patients suffering from hereditary cerebral hemorrhage associated with amyloidosis Dutch type (HCHWA-D). Amyloid beta peptides are fragments of the precursor protein beta-amyloid (APP) that contains different number of amino acids, typically 38-43 amino acids.

Amyloid beta peptides are usually referred to as "includes AB (x-y)" where x is the number of amino acids at the amino-end of amyloid beta-peptide, and y means the number of amino acids at the carboxy end. So, for example, includes AB(1-40) is an amyloid beta peptide, the amino-end of which begins in position 1 of the amino acid sequence and the carboxy-end of ends � position 40 the amino acid sequence, where the specified sequence represented by SEQ ID NO:l. Examples of amyloid beta-peptides, which may be determined by the method according to the invention, include, but are not limited to, includes AB(1-38) (SEQ ID NO:2), includes AB(1-39) (SEQ ID NO:3), includes AB(1-40) (SEQ ID NO:1), includes AB(1-41) (SEQ ID NO:4), includes AB(1-42) (SEQ ID NO:5), includes AB(1-43) (SEQ ID NO:6), includes AB(11-42) (SEQ ID NO:7), includes AB(3-40) (SEQ ID NO:8), includes AB(3-42) (SEQ ID NO:9), includes AB (4-42) (SEQ ID NO:10), includes AB(6-42) (SEQ ID NO:11), includes AB(7-42) (SEQ ID NO:12), A13(8-42) (SEQ ID NO:13), includes AB(9-42) (SEQ ID NO:14), includes AB(x-40), Includes AB(x-42) and includes AB (x-38), all amyloid beta peptides, which belong to many species of amyloid beta-peptides, where the individual species are unidentified. In preferred embodiments of the invention, amyloid beta-peptides that can be detected by the method according to the invention are these AB(1-40) and includes AB(1-42).

As used herein, the term "includes AB(1-42)" means a peptide which consists of 42 amino acids corresponding to amino acids 672-713 (SEQ ID NO:5) APP, which is produced by sequential proteolytic cleavage of the precursor protein of amyloid protein (SEQ ID NO:15) under the action of β - and γ-secretase.

As used herein, the term "includes AB(1-40)" means a peptide which consists of 40 amino acids corresponding to amino acids 672-711 (SEQ ID NO:1), and which is produced by sequential proteolytic cleavage of the precursor protein of amyloid protein (SEQ ID NO:15) under the action of β - and γ-secretase.

Used in n�standing the invention, the term "biological sample" includes (1) a biological fluid, such as whole blood, serum, plasma, urine, lymph, saliva, semen, sputum, tears, mucus, sweat, milk, brain extracts and cerebrospinal fluid; (2) blood components such as plasma, serum, blood cells, and platelets; (3) extracts obtained from solid tissues or organs such as the brain; and (4) the extracts obtained from cultures of cell lines or primary cells, human or animal, such as primary human neurons and primary neurons isolated from transgenic mice bearing genes APP, such as cells PDAPP isolated from a transgenic animal (e.g., mouse), as well as the cell line of human kidney 293 cell line neuroglioma human cell line HeLa human primary endothelial cell line (e.g., HUVEC cells), primary human fibroblasts or primary line of lymphoblasts (including endogenous cells, derived from patients with APP mutations), mixed primary cell culture of the human brain, including neurons, astrocytes and neuroglial cells) or the cell line is Chinese hamster ovary (CHO). The methods according to the invention are particularly suitable for the measurement of these AB in the blood sample of a human or animal, not human, such as whole blood, plasma or sample containing any of the components of CRO�and in any quantity.

The term "whole blood" means blood taken from a human or animal containing the cell components and liquid components. Whole blood may be stored in a coiled condition or not in a coiled condition. The term "whole blood" includes blood, in which a portion of the cellular components or all cellular components, such as leukocytes or red blood cells were subjected to lysis.

The term "plasma" means the liquid component of whole blood. Depending on the applied method of separation in the plasma can be completely absent cellular components or plasma may contain a different number of platelets and/or small amounts of other cellular components.

The term "serum" refers to the plasma that lacks a clotting factor, such as a protein fibrinogen, and other coagulation factors.

Used here, the term "free amyloid beta peptide" means amyloid beta peptides that are not associated with any component of the biological sample and which are readily available for binding to specific antibody. This peptide may be determined by standard immunological methods by contacting the biological sample with an antibody specific to a given peptide. In a preferred embodiment of the invention, the level� free amyloid peptide determined in plasma.

As used herein, the term "amyloid beta-peptide associated with macromolecular components" means amyloid beta-peptide, which ecovalence is associated with molecules or attached to molecules present in the analyzed biological sample. This peptide is normally inaccessible for immunological detection, and therefore for separation of the peptide from components require pre-treatment of such a biological sample. In these conditions, amyloid beta peptide associated to macromolecular components, will be released from these components and will be available for immunological detection of specific antibodies. Because biological sample already contains some amount of free amyloid beta-peptide, the total amount of free amyloid peptide after contacting the sample with a protein-solubilizers agent will include a certain level aggregates such is usually the presence of free amyloid beta-peptide and amyloid beta-peptide released after treatment of protein-solubilizers agent. If you need to determine the level of amyloid beta peptide associated to macromolecular components in biological sample, it can be done put�m determine the level of free amyloid beta-peptide prior to treatment of protein-solubilizing agent and the level of free amyloid beta-peptide after treatment with protein-solubilizing agent and subtraction of the first magnitude from the second magnitude. In order to implement the present invention generally define the aggregated level of free amyloid beta-peptides, which includes the level usually present free of amyloid beta-peptides and the level of amyloid beta peptides released from the macromolecular components after the processing of protein-solubilizers agent. Therefore, the parameter that is usually determined in the case of sample processing to amyloid peptide dissociation of macromolecular components, represents the sum of the amount of free peptide present in the sample, and the peptide is associated with macromolecular components.

Macromolecular components of the sample that can bind to amyloid beta peptides and that can be a pool of amyloid beta peptides associated with macromolecular components include both proteins and peptides. In the specific case of the implementation of this method on samples of blood or plasma macromolecular components include, but are not limited to, proteins, and blood lipids. Representative blood proteins are albumin, immunoglobulin G, immunoglobulin E, immunoglobulin M, immunoglobulin A, fibrinogen (fibrin and products of their decomposition), antitrypsin alpha-1, prealbumin, acid glycoprotein al�a-1, fetoprotein, alpha-1, haptoglobin alpha-2 macroglobulin, ceruloplasmin, transferrin, C3/C4-beta-2-microglobulin, beta lipoprotein, alpha-, beta - and gamma-globulins, C-reactive protein (CRP), prothrombin, thyroxine-binding protein, transthyretin, etc. Representative blood lipids are free fatty acids, cholesterol, triglycerides, phospholipids, sphingolipids, etc. the Amount of amyloid beta peptide associated to macromolecular components can be determined by contacting the acellular biological sample with a protein-solubilizers agent under conditions suitable to induce the release of these amyloid beta-peptides of macromolecular components.

In the present description, the term "contacting" means adding to the sample a sufficient amount of a solution containing protein-solubilizers agent, such that the concentration of protein-solubilizing agent in the mixture was sufficient for effective solubilization of amyloid beta-peptide, which binds to proteins and cells in the sample. In this case, preferably, the specified protein-solubilizers agent is present in a buffer solution in an amount which would not cause significant changes in pH of the sample.

Used here� the term "protein-solubilizers agent" means any compound of definite composition, capable of altering the secondary, tertiary and/or Quaternary structure of the polypeptide while maintaining their primary structure. As for the properties of these protein-solubilizing agents, the agents have the ability to increase the solubility of proteins in the sample and to prevent intermolecular and intramolecular aggregation of proteins. Protein-solubilizers agents suitable for use in the present invention include, but are not limited to, detergents, chaotropes agents, reducing agents, or mixtures thereof.

Used here, the term "detergent" is generally synonymous with the term "surfactant" means amphipathicity surface-active substances which, when added to a liquid, reduces the surface tension in the liquid compared to the surface tension the same liquid in which the surfactant is absent. Detergents can also prevent protein aggregation and non-specific interaction or binding with impurities contaminating protein of interest. Detergents suitable for use in the present invention include, but are not limited to, nonionic (neutral), anionic, cationogenic or zwitterionic detergents.

Examples of nonionic or neutral deterg�the components are, but are not limited to, detergents series tween, such as tween® 20, tween® 21, tween® 40, tween® 60, tween® 61, tween® 65, tween® 80, tween® 81, tween® 85, detergents series Span®, such as Span® 20; detergents series targetrow, such as tergitol type 15-S-12; detergents of the series Brij®, such as Brij® 35, Brij® 56, Brij® 72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P; detergents series tween, such as tween® 20, tween® 21, tween® 40, tween® 60, tween® 61, tween® 65, tween® 80, tween® 81, tween® 85; detergents Triton series®, such as Triton® X-100, Triton® X-114, Triton® CF-21, Triton® CF-32, Triton® DF-12, Triton® DF-16, Triton® GR-5M, Triton® X-102, Triton® X-l5, Triton® X-151, Triton® X-207, Triton® X-165, Triton® X-305, Triton® X-405, Triton® X-45, Triton® X-705-70, or non-ionic conservative variant of at least one of the detergents.

Examples of anionic detergents include, but are not limited to, cholic acid and its derivatives, tauroholeva acid, Triton X-200, Triton W-30, Triton-30, Triton-770, dioctylsulfosuccinate, N-oxide (N5N-dimethyldodecylamine, 1-sulfonates, sodium N-lauroylsarcosine or salts of fatty acids.

Examples cationogenic detergents include, but are not limited to, mono - and dimethylamine fatty acids, salts of alkyltrimethylammonium, salt dialkyldimethylammonium, of the alkylamine acetates, acetates of trialkylamine, salt alkyldimethylbenzylammonium, salt dialkyldimethylammonium�I, the halide of alkylpyridine and salt alkyl(alkyl-substituted)pyridinium, salt alkyltrimethylenedi, salt alkilaminometilen, salt alkylquinoline, salt alkylenediamine, salts of N,N-alkylmethacrylamide, salt 1,1-dialkylamide, salts of 4,4-dealkilirovania, salt 1-oxide 4,4-dealkilirovania, metilsulfate methyl-bis(alkylated)-2-alkylimidazole (and other salts), metilsulfate methylbis(acylaminoalkyl)-2-hydroxyethylamine (and other salts), salts of alkylamide-dimethylbenzylamine, salt carboxyethyl-alkyldimethylammonium, alkylamine oxides, oxides of alkyldimethylammonium, salts of poly(vinylpyrrolidone), salts of poly(vinylpyridine), polyethyleneimine, bicarbonates trialkylphosphine (and other salts), salts trialkylphosphine, salt alkylarylsulfonate and salt alkyldimethylammonium.

Examples zwitterionic detergents include, but are not limited to, 3-[(3-cholamidopropyl)dimethylamine]-1-propanesulfonate (CHAPS); 3-[(3-cholamidopropyl)dimethylamine]-2-hydroxy-1-propanesulfonate (CHAPSO), N-(alkyl C10-C16)-N,N-dimethylglycine-betaine (EMPIGEN BB); caprolactamate (SB3 - 10); 3-[N,N-dimethyl(3-myristoylation)ammonia]propanesulfonate (amidosulfuron-14; ASB-14); N-tetradecyl-N,N-dimethyl-3-ammonia-1-propanesulfonate (3-14-detergent; ZWITTERGENT); N-dodecyl-N,N'-dimethyl-3-ammonia-1-propanesulfonate; N-octadecyl-N,N-dimethyl-3-ammonia-1-�propansulfonate; N-decyl-N,N-dimethyl-3-ammonium-1-propanesulfonate; mirataine CB; mirataine BB; mirataine CBR; mirataine ACS; Miramar 2MHT and Miramar 2MCA.

In a preferred embodiment of the invention, protein-solubilization reagent is a detergent. In an even more preferred embodiment of the invention, the specified detergent is tween 20. In an even more preferred embodiment of the invention, tween 20 was used at a concentration of 0.5%.

As used herein, the term "chaotropic agent" means a compound or mixture of compounds, which decompose hydrogen bonds and disrupt hydrophobic interactions between proteins and proteins inside. Chaotrope agents used in high concentrations, disrupt the secondary structure of the protein and form proteins in solution, which are not soluble. Suitable chaotrope agents include, but are not limited to, urea, isothiocyanate, guanidine, sodium thiocyanate (NaSCN), guanidine-HCl, chloride guanidine, thiocyanate guanidine, tetrachlorinated lithium, sodium perchlorate, tetrachlorinated rubidium, potassium iodide or triptorelin cesium.

Used here, the term "reducing agent" means any compound or material which maintains sulfhydryl groups in the reduced state and restores intramolecular or intermolecular disulfide bonds. So, for example, appeared�a nd, suitable for use in the method according to the invention, are sulfhydryl or phosphine reductants. Examples of sulfhydryl reducing agents are dithiothreitol (DTT), dithioerythritol (DTE) and β-mercaptoethanol. Examples of phosphine reductants are tributylphosphine (TBP) and Tris-carboxyhemoglobin (TCEP).

Typically, the biological sample is first treated to remove cellular fraction. Then the acellular sample is subjected to contacting with protein-solubilizers agent. In a preferred embodiment of the invention, the sample is diluted with buffer containing protein-solubilizers agent. Usually a sample of 5-fold diluted in buffer solution containing tween 20.

As used herein, the term "buffer solution" means any substance or mixture of compounds in solution that have the ability to neutralize acids and bases, but it does not have any significant impact on the original acidity or basicity of a solution. Suitable buffer solutions used in the method according to the invention, include, but are not limited to, Tris buffer solution, phosphate buffer solution, borate-buffer solution, carbonate buffer solution, the buffer solution containing glycine-sodium hydroxide, etc. Preferred buffer solution is fosfat�-buffer solution, such as phosphate buffered saline or PBS.

The amount of solution containing protein-solubilizers agent, which is added to the biological sample is not critical provided that will be achieved at a sufficient level of dissociation of the amyloid beta-peptide. For example, the biological fluid may be diluted in a solution containing a protein-solubilizers agent at a dilution of at least 1/2 (about./vol.), 1/3 (about./vol.), 1/4 (about./vol.), 1/5 (about./vol.), 1/6 (about./vol.), 1/7 (about./vol.), 1/8 (about./vol.), 1/9 (about./vol.), 1/10 (about./vol.), 1/20 (about./vol.), 1/50 (about./vol.), 1/60 (about./vol.), 1/80 (about./vol.), 1/90 (about./vol.), 1/100 (vol./about.) or more. The experts know that can be used any combination of the protein-solubilizing agents specified in the dilution provided that the final concentration of protein-solubilizing agent will be adequate to achieve the desired effect. For example, a solution containing a protein-solubilizers agent, can include specified(e) selected(e) protein-solubilizers(e) agent(s) in a concentration of from 0.001% to 0.5% (wt./vol.). After dilution in the specified solution containing protein-solubilizers agent specified biological fluid typically contains the specified(s) surfactant(s) substance(s) in a concentration of less than 0.1% (wt./vol.), preferably, less than 0.6% (wt.vol.), more preferably, not more than 0.5% (wt./vol.), most preferably, no more than 0,45% (wt./about.) and even more preferably, of 0.5%.

Buffer systems suitable for use in the present invention are Tris-HCl buffers, including salt, such as NaCl or KCl, and optional, BSA. Specific buffer systems include, but are not limited to:

50 mm Tris-HCl, pH 8, 0.5 M NaCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 0.5 M NaCl, and 0.05%; BSA, 0.05% tween 20, 1M GuHCl;

50 mm Tris-HCl, pH 8, 0.5 M KCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 0.5 M KCl, and 0.05%; BSA, 0.05% tween 20, 1M GuHCl;

50 mm Tris-HCl, pH 8, 0.5 M NaCl, and 0.05%; BSA, 0.05% tween-80;

50 mm Tris-HCl, pH 8, 0.5 M KCl, and 0.05%; BSA, 0.05% tween-80;

50 mm Tris-HCl, pH 8, 0.5 M NaCl; and 0.05%; BSA, 0.05% of Triton X-100

50 mm Tris-HCl, pH 8, 0.5 M KCl, and 0.05%; BSA, 0.05% of Triton X-100;

50 mm Tris-HCl, pH 8; 0,05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 0.5 M NaCl, 0,1%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 0.5 M NaCl, and 0.05%; BSA, 0.1% tween-20;

50 mm Tris-HCl, pH 8, 0.5 M NaCl, 0,1%; BSA, 0.1% tween-20;

50 mm Tris-HCl, pH 8, 1M NaCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 1.5 M NaCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 2M NaCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 2.5 M NaCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 3M NaCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 8, 0.5 M NaCl, and 0.05%; BSA, 0.05% tween 20, 10% DMSO;

50 mm Tris-HCl, pH 8, 0.5 M NaCl, and 0.05%; BSA, 0.05% tween 20, 0.5 M GuHCl;

50 mm Tris-HCl, pH 6, 0.5 M NaCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 7, 0.5 M NaCl, and 0.05%; BSA, 0.05% tween 20;

50 mm Tris-HCl, pH 9, 0.5 M NaCl, and 0.05%; BSA, 0.05% TV�n-20;

50 mm Tris-HCl, pH 8, 0.5 M NaCl, and 0.05%; BSA.

For example, if the protein-solubilizing agent used tween 20, then, preferably, its concentration is 0,004-0,02%, and more preferably of 0.005-0.01% (wt./vol.).

Stage contacting is preferably carried out at low temperature to inhibit the activity of proteolytic enzymes present in the sample. Suitable temperatures are a temperature of about 0-10°C, preferably about 3-5°C, for example about 4°C.

After contacting the biological fluid with a solution containing a protein-solubilizers agent, the two liquids can be mixed. Mixing may be effected by stirring, preferably, shaking, more preferably, high-speed shaking, and most preferably, vortex shaking for at least 5 seconds, preferably for at least 10 seconds, and more preferably at least 15 seconds (for example, 15-50 seconds). The preferred speed of the specified mixing, stirring, shaking, high-speed shaking or vortex shaking is at least 250 rpm, preferably at least 500 rpm, more preferably at least 1000 rpm, and most preferably, at�Erno 2000-2500 rpm.

The step of contacting is performed under conditions suitable to achieve partial or preferably complete dissociation of amyloid beta-peptide from protein and lipids present in a biological sample. Such conditions can be adequately determined by an average person skilled in the art by monitoring the amount of amyloid beta-peptide that can be detected before the stage of contacting, and which gradually increase in different periods of time after the step of contacting. The experiment can be determined, as described in the example presented in the experimental part.

The person skilled in the art it is known that if the level of amyloid beta-peptide is determined by diluting a biological sample buffer containing protein-solubilizers agent, the level of free amyloid beta-peptide, determined by immunoassay, it is adjusted with the factor used previously dilutions of a biological sample.

Thus, in a preferred embodiment of the invention, a parameter defined at the stage (i) of the method according to the invention are one or more parameters selected from the group consisting of levels of free ABETA40 peptide in a biological sample taken from a specified individual�of mind (hereinafter referred to 1ab40 or UP includes AB(1-40)), the aggregate level of free ABETA40 peptide in the biological sample, and ABETA40 peptide associated with the components of the specified biological sample obtained as described above (hereinafter referred to 2ab40 or DP includes AB(1-40)), levels of free ABETA42 peptide in a biological sample (hereinafter referred to 1ab42 or UP includes AB(1-42)), levels and units of the free ABETA42 peptide in the biological sample, and ABETA42 peptide associated with the components of the specified biological sample obtained as described above (hereinafter referred to 2ab42 or DP includes AB(1-42)).

As used herein, the term "amyloid beta-peptide-associated cells" means amyloid beta-peptide, which ecovalence is associated with the surface of cells present in a biological sample, and which is not able to antibodies added to the sample, and therefore it is immunological netdetective. Usually, if the biological sample is blood, amyloid beta-peptide associated with erythrocytes, leukocytes, including neutrophils, eosinophils, basophils, lymphocytes and monocytes, and platelets.

Can be determined the amount of amyloid beta peptide associated to cells in the sample, and this value may be used alone or in combination with other parameters related to amyloid beta-peptide�m, used in the methods according to the invention. For this purpose it is first necessary to isolate the cellular fraction from a biological sample. This can be carried out using any method known in the art, such as centrifugation, sedimentation, filtration, etc. After separation of the cellular fraction from a biological sample, the cells are contacted with the protein-solubilizers agent.

Suitable protein-solubilizers agents are detergents, chaotrope agents and reducing agents as defined above, and is usually used in a buffer solution at an appropriate concentration. Suitable agents, buffer solutions, and the concentration of agents in the buffer solution described above. Stage contacting is carried out mainly as described above in method release of amyloid peptide that is associated with the components (proteins and lipids) of the biological sample. In a preferred embodiment of the invention, the specified protein-solubilizers agent is a detergent. In an even more preferred embodiment of the invention, the specified detergent is tween 20. Suitable concentrations of tween 20 used as protein-solubilizing agent, identified above and are of 0.004 to 0.02%, and more preferably of 0.005-0.01% (wt./vol.).

Stage contacting is preferably carried�tlaut at low temperature to inhibit the activity of proteolytic enzymes, present in a sample. Suitable temperatures range from about 0-10°C, preferably about 3-5°C, for example about 4°C.

Typically, the step of contacting is carried out by resuspension of the cell fraction in a biological sample with a solution containing a protein-solubilizers agent. Specified the resuspension can be achieved by careful pipetting by abstraction and release, by mixing, preferably, shaking, more preferably, high-speed shaking, and most preferably, vortex shaking for at least 5 seconds, preferably for at least 10 seconds, and more preferably at least 15 seconds (for example, 15-50 seconds). The preferred speed of the specified mixing, stirring, shaking, high-speed shaking or vortex shaking is at least 250 rpm, preferably at least 500 rpm, more preferably at least 1000 rpm, and most preferably, about 2000-2500 rpm.

The step of contacting is performed under conditions suitable to achieve partial or preferably complete dissociation of amyloid beta-peptide from cells present in a biological sample. Such conditions can be adequately ODA�divided by the average person skilled in the art by monitoring the amount of amyloid beta-peptide, which can be detected before the stage of contacting, and which gradually increase in different periods of time after the step of contacting. The experiment can be determined, as described in the example in the experimental section.

Thus, in a preferred embodiment of the invention, a parameter defined at the stage (i) of the method according to the invention are one or more parameters selected from the group consisting of ABETA40 level associated with cells present in a biological sample (denoted hereinafter 3ab40 or ST includes AB(1-40)), and the level of ABETA40 associated with cells present in a biological sample (hereinafter referred to 3ab42 or ST includes AB(1-42)).

The following stage of the method according to the invention involves comparing the value of at least one of the parameters (b) or (c) or the value of the parameter calculated by the arithmetic combination of one or more parameters (a) to(c) with a reference value corresponding to the value of the specified parameters (b) or (c) the calculated or specified parameter in the reference model, where (a), (b) and (c) is defined, as described above, and correspond to the level of free amyloid beta-peptide in the biological sample of the individual, respectively (a)the levels of aggregates free�defined amyloid peptide in the biological sample, taken from a specified individual, and levels specified amyloid beta-peptide-associated macromolecular components present in the specified biological sample (b), and the level of amyloid beta peptide associated to cells in a biological sample taken from a specified individual (c).

The parameters used in stage (ii) of the method according to the invention are any direct parameters, i.e., parameters that can be determined by the direct method described above, and which correspond to the parameters previously identified as 2ab40, 3ab40, 2ab42 and 3ab42. Alternatively, the calculated parameter can be also obtained by an arithmetic combination of one or more direct options. Practically, the calculated parameters with diagnostic value can be obtained by any arithmetic combination of two or more parameters, including addition, subtraction, multiplication, division and combinations thereof. Specific calculated markers used in the method according to the invention, include, but are not limited to, 2ab40/2ab42, 3ab40/3ab42, 2ab40/3ab40, 2ab42/3ab42, 1ab40+2ab40, 1ab40+3ab40, 2ab40+3ab40, 1ab40+2ab40+3ab40, 1ab42+2ab42, 1ab42+3ab42, 2ab42+3ab42, 1ab42+2ab42+3ab42, 1ab40+2ab40+1ab42+2ab42, 1ab40+3ab40+1ab42+3ab42, 2ab40+3ab40+2ab42+3ab42, 1ab40+2ab40+3ab40+1ab42+2ab42+3ab42, (1ab40+2ab40)/(1ab42+2ab42), (1ab40+3ab40)/(1ab42+3ab42), (2ab40+3ab40)/(2ab42+3ab42), (1ab40+2ab40+3ab40)/(1ab42+2ab42+3ab42), (1ab42+2ab42)/(1ab40+2ab40), (1a42+3ab42)/(1ab40+3ab40), (2ab42+3ab42)/(2ab40+3ab40), (1ab42+2ab42+3ab42)/(1ab40+2ab40+3ab40), 2ab40-1ab40, 2ab42-1ab42 and (2ab40-1ab40)/(2ab42-1ab42). In a preferred embodiment of the invention, the calculated parameter obtained by adding parameters 2ab40 and 3ab40 (hereinafter referred to T40). In another preferred embodiment of the invention, the calculated parameter obtained by adding parameters 2ab42 and 3ab42 (hereinafter referred to T42). In yet another preferred embodiment of the invention, the calculated parameter obtained by adding parameters 2ab40, 3ab40, 2ab42 and 3ab42 (hereinafter referred Τ-βΑΡΒ).

As used herein, the term "reference value" means the value of the parameter that is used for comparison and which was identified in an individual not suffering from a neurodegenerative disease, or an individual who had no history of any neurodegenerative disease. Preferably, individuals, who provided reference values for various parameters and the calculated parameters are patients, who had no complaints of memory loss were observed normal behavior, as indicated by neuropsychological tests, and a lack of structural changes on MRI.

In particular, were chosen as reference values, which corresponded to a sensitivity above 85% and specificity above 75%. In another preferred embodiment of the invention, reference ve�Iceni are selected so that to match the higher sensitivity 70% and specificity above 70%. Preferably, the reference value is difficult to estimate with accuracy of at least 80%.

At the stage (iii) of the method according to the invention, the diagnosis of neurodegenerative diseases, the determination of the stage prior to a neurodegenerative disease, or differentiation of neurodegenerative disease from a stage prior to a neurodegenerative disease is carried out using a specific series of markers or calculated markers, which provide adequate levels of specificity and sensitivity. Thus, in a specific example, the diagnosis of neurodegenerative diseases pose by comparing the value of the parameter selected from the group consisting of 3ab40 and 2ab42, or the amount calculated parameter selected from the group consisting of 2ab40+3ab40 and 2ab40+3ab40+2ab42+3ab42.

In another specific embodiment of the invention, detection of the disease stage prior to a neurodegenerative disease is carried out by comparing the value of the parameter selected from the group consisting of 2ab40, 3ab40 and 2ab42, or the amount calculated parameter selected from the group consisting of 2ab40+3ab40, 2ab40+3ab40+2ab42+3ab42, 1ab40+2ab40+3ab40+1ab42+2ab42+3ab42, 1ab40+2ab40+3ab40, 1ab42+2ab42+3ab42, 1ab40+1ab42+2ab42+3ab42, 1ab40+2ab40+1ab42+2ab42 and 1ab40+3ab40+1ab42+3ab42.

In another embodiment, Fig�plants, differentiation of neurodegenerative disease from a stage of disease prior to the specified neurodegenerative disease is carried out by comparing the value of the parameter selected from the group consisting of 3ab40 and 2ab42, or by comparing values of the calculated parameter selected from the group consisting of 2ab40+3ab40, 2ab40+3ab40+2ab42+3ab42 and 1ab40+2ab40+1ab42+2ab42.

Adequate reference values used in various diagnostic methods according to the invention, are systematized in table 1.

Table 1
Systematic parameters and thresholds for preferred methods according to the invention
MethodThreshold value (PG/ml)
The detection of a stage prior to a neurodegenerative disease2ab40/DP(Αβ1-40)63,8
The diagnosis of neurodegenerative diseases3ab40/CB(Αβ1-40)71,9
The detection of a stage prior to a neurodegenerative disease3ab40/CB(Αβ1-40) 71,1
Differentiation of neurodegenerative3ab40/CΒ(Αβ1-40)To 211.3
disease from a stage preceding the specified neurodegenerative disease
The diagnosis of neurodegenerative diseases2ab42/DP(Αβ1-42)47,4
The detection of a stage prior to a neurodegenerative disease2ab42/DP(Αβ1-42)50,3
Differentiation of neurodegenerative disease from a stage preceding the specified neurodegenerative disease2ab42/DP(Αβ1-42)Of 151.7
The diagnosis of neurodegenerative diseases3ab42/CB(Αβ1-42)76,9
The detection of a stage prior to a neurodegenerative disease3ab42/CB(Αβ1-42)58,8
The diagnosis of neurodegenerative diseases2ab40+3ab40/T40132,7
The detection of a stage prior to a neurodegenerative disease2ab40+3ab40/T40132,7
Differentiation of neurodegenerative disease from a stage preceding the specified neurodegenerative disease2ab40+3ab40/T40550,8
The diagnosis of neurodegenerative diseases2ab42+3ab42/T42115,8
The detection of a stage prior to a neurodegenerative disease2ab42+3ab42/T42103,3
The diagnosis of neurodegenerative diseases2ab40+3ab40+2ab42 +3ab42/T-β235,5
The detection of a stage prior to a neurodegenerative disease2ab40+3ab40+2ab42 +3ab42/T-β235,5
Differentiation of neurodegenerative disease from a stage preceding the specified neurodegenerative disease2ab40+3ab40+2ab42 +3ab42/T-β778,1
Detection of PT�dia, prior neurodegenerative disease1ab40+2ab40+3ab40+ 1ab42+2ab42+3ab42272,1
The detection of a stage prior to a neurodegenerative disease1ab40+2ab40+3ab40155,8
The detection of a stage prior to a neurodegenerative disease1ab42+2ab42+3ab42124,3
The diagnosis of neurodegenerative diseases1ab40+2ab40+1ab42 +2ab42158,3
The detection of a stage prior to a neurodegenerative disease1ab40+2ab40+1ab42 +2ab42's 142.4
Differentiation of neuro-degenerative diseases from the stage preceding the specified neurodegenerative disease1ab40+2ab40+1ab42 +2ab42161,2
The detection of a stage prior to a neurodegenerative disease1ab40+3ab40+1ab42 +3ab42154,7

After determining the value of the parameter or calculated parameter diagnostics of neurodegenerative�th disease, the detection of a stage prior to a neurodegenerative disease, or differentiation of neurodegenerative disease from a stage preceding the specified neurodegenerative disease according to the invention, can be achieved if there is a change in the parameter value or values of the calculated parameter is compared with a reference value.

The term "modification" means a statistically significant increase or decrease in the value of the considered parameter is compared with a reference value.

As used herein, the term "statistically significant" refers to statistical probability analysis, which is a non-random combination of two or more of the outcomes, endpoints or outcomes of the disease, that is, a certain degree of mathematical accuracy, that the parameter value associated with a specific group of patients corresponds to the reference value.

Statistically significant variation in values can be determined using p-values. For example, when using p-value, the parameter is identified as a statistically significant variation, if the p-value is less than 0.1, preferably less than 0.05, more preferably, less than 0.01, even more preferably less than 0.005, and the most preferred�Thelen, less than 0.001.

Usually, the value of the parameter in question can be considered "high" if the specified value is at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more higher than the reference value. On the other hand, the value of the parameter can be considered "low" if the specified value is at least 0.9 times, 0.75 times, 0.2 times, 0.1 times 0.05 times 0,025 in times of 0.02 times, 0.01-fold, 0.005-fold or less below the reference value. In a particular embodiment of the invention, a change in the magnitude of the parameter or the values of the calculated parameter is compared with a reference quantity is increased to the specified value.

In the present invention can be applied to any method suitable for the determination of peptides. For example, the concentration of amyloid beta-peptide can be determined using one or more techniques chosen from Western blot analysis, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), analysis by surface plasmon resonance, reaction with precipitins, immunodiffusion analysis by diffusion in the gel, radioimmunoassay (RIA), cell sorting with activation of fluorescence (FACS), two-dimensional gel electrophoresis, capillary electrophoresis, mass spectroscopy (MC), matr�: laser desorption/ionizing time-of-flight MS (MALDI-TOF), laser desorption ionization-time of flight on an activated surface (SELDI-TOF), high performance liquid chromatography (HPLC), liquid Express chromatography of proteins (IAHB), multidimensional liquid chromatography (MGH), tandem mass spectrometry (MS/MS), thin layer chromatography, analysis by the method of expression on the protein chip and laser densitometry.

In a preferred embodiment of the invention, the determining of at least one or more 1ab40, 1ab42, 2ab40, 2ab42, 3ab40 and 3ab42 is carried out by immunoassay. As used herein, the term "immunological method", if it refers to the definition, means any method that involves using one or more antibodies specific to the substance of the target to determine the amount/concentration of the specified substance target to the exclusion of other substances present in the sample. Suitable immunological methods include, but are not limited to, Western blot analysis, immunoprecipitation, solid-phase enzyme-linked immunosorbent assay (ELISA) analysis by surface plasmon resonance, a radioimmunoassay (RIA).

Specialists in this field it is obvious that the implementation of methods immunological detection according to the invention can be used an antibody of any type, provided that such antibody to buttonlight specificity, sufficient for effective differentiation of molecules of amyloid beta-peptide in the sample from other substances. Molecules of antibodies suitable for use in immunological methods according to the invention, include, but are not limited to:

(i) "intact" antibody containing the antigen-binding variable region and a constant domain of the light chain (CL) and constant domains of the heavy chain CHl, CH2 and CH3,

(ii) "Fab"fragments obtained by hydrolysis of an intact antibody with papain and containing a single antigen-binding site and a CL region and CH1,

(iii) "F(ab')2"fragments obtained by hydrolysis of intact antibodies by pepsin and containing two antigen-binding site,

(iv) "Fab"fragments, containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain and only one antigen-binding site. Fab'fragments differ from Fab fragments by the fact that attached to them a few residues at the carboxy-end of the CH1 domain of the heavy chain, including one or more cysteines, derived from a hinge region of the antibody.

(v) "Fv" is the minimum antibody fragment that contains a full-sized site of antigen recognition and antigen-binding site. This region consists of a dimer composed of one variable domain of the heavy chain and one �arabellege domain of light chain, firmly connected with each other by non-covalent bond. This configuration represents the three hypervariable region (CDRs) of each variable domain, which form a specific antigen-binding site on the surface of the dimer VH-VL. Overall, the six hypervariable regions report to the antibody binding specificity to the antigen. However, even a single variable domain (or half of Fv containing only three hypervariable region that is specific for an antigen) has the ability to recognize the antigen, and contacting the antigen, although with lower affinity than the full-sized binding site.

(vi) Single-chain fragments of the Fv or "scFv" antibody containing domains VL and VH of antibodies, where these domains are present in a single polypeptide chain. Preferably, a VL region and VH are connected by a polypeptide linker, which allows the fragment scFv to form the desired structure suitable for binding with the antigen.

(vii) "Dentical" containing the variable domain of the heavy chain (VH) connected to the variable domain light chain (VL) on the same polypeptide chain (VH-VL) by a peptide linker that is too short to form a pair between the two domains on the same chain. This provides for mating with complementary domains of another chain and stimulates Tue�, ar dimeric molecule with two functional antigen-binding sites.

(viii) "Bespecifically antibodies (BAb), which represent a bivalent single-chain antibodies (or their effective immunotherapeutic fragments with two antigen-binding site with different specificity. These two antigen-binding site may be linked to each other by a chemical method or methods of genetic engineering well known to the experts.

All these fragments of antibodies may also be modified by standard methods, known in the art, for example, using the amino acid(s) deletion(s), insertion(s), substitutions (replacements), addition(s) and/or recombination (and/or any(s) other(s) modification(s) (for example, a known post-translational and chemical modifications such as glycosylation and phosphorylation), both separately and in combination. Methods for introducing such modifications in the DNA sequence encoding the corresponding amino acid sequence of the immunoglobulin chain are well known in the art; see, for example, manual Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2ndedition 1989 and 3rdedition 2001.

Antibodies can be polyclonal and monoclonal antibodies. To obtain polyclonal antibodies, various hosts including goats, rabbits, rats, mice, camels one humped camels, llamas, humans, birds,etc., can be immunitary by injection of the peptide, corresponding to the fragment of Αβ40 or Αβ42 having immunogenic properties. Depending on the type of the host to enhance the immune response can be used in different adjuvants. Such adjuvants include, but are not limited to, adjuvant freind, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used to introduce a person, especially preferred are BCG (bacille Calmette-Guerin) andCorynebacterium parvum. If the antigen is a peptide, it can be used for conjugation to a protein that is immunogenic for immunogenic species. For example, the antigen can be conjugated with keyhole lymph snails (KLH), Blue Carrier (keyhole isolated fromConcholepas concholepas), bovine thyroglobulin or soybean trypsin inhibitor by means of bifunctional or derivatizing agent, such as (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic acid anhydride or SOCl2.

For producing monoclonal antibodies can be applied standard methods. So, for example, monoc�national antibodies can be obtained by a hybrid method first described by Kohler et al., Nature, 256:495 (1975), in accordance with the procedure described in detail in sections 11.4-11.11 guide Ausubel, F. M. et al. (Current Protocols in Molecular Biology, John Wiley & Sons Inc; ring-bound edition, 2003). Alternatively, monoclonal antibodies can be isolated by methods of recombinant DNA from phage libraries of antibodies obtained by the methods described in McCafferty et al., Nature, 348:552-554 (1990). In the works Clacksoii et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol, 222:581-597 (1991) described the allocation of murine and human antibodies, respectively, using phage libraries. Other publications describe the production of high-affinity (nm range) human antibodies by moving the chains (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection andin vivorecombination used as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids. Res., 21:2265-2266 (1993)). Thus, these methods provide a viable alternative to traditional hybrid technology of producing monoclonal antibodies.

Polyclonal antibodies can be used directly as antisera obtained from immunogenic owners after blood collection and removal of fibrin clot. Monoclonal antibodies can be used directly as hybrid supernatant of culture and�and as ascitic fluid after implantation of hybridomas into the abdominal cavity of a suitable owner. Alternatively, the immunoglobulin molecule as polyclonal and monoclonal, can be purified by standard methods, such as affinity purification using peptides derived from amyloid beta-peptide clearance in sedentarism gel, cleaning methods, HPLC or RP-HPLC, purification using size exclusion chromatography purification on a column of protein A, or any combination of these methods.

Suitable antibodies that can be obtained by immunological methods according to the invention, include, but are not limited to:

(i) Antibodies that recognize the region, starting from the N-terminal region of amyloid beta-peptides, such as antibodies specific to epitopes localized in the provisions of amino acids 1-16, 1-17, 13-28, 15-24, 1-5 and 1-11 peptides Αβ40 or Αβ42; and antibodies produced against the peptide corresponding to the C-terminal region of Αβ42 peptide, which binds specifically with Αβ42 and not enter into any explicit cross-react with Αβ40 or Αβ43,

(ii) Antibodies produced against the peptide corresponding to the C-terminal region of Αβ40 peptide that specifically bind to Αβ40 and not enter into any explicit cross-react with Αβ42, Αβ39, Αβ38, Αβ41 or Αβ43,

(iii) Antibodies that simultaneously recognize the C-terminal region Αβ40 and Αβ42,

(iv) Anti�La, which are specific to the area of the intersection of amyloid beta-peptides, and which can be used to identify amyloid beta-peptides from other APP fragments, and localized in the provisions of amino acids 16 and 17 amino acid sequence, usually extending from amino acid residues 13 to amino acid residues 28,

(v) a Combination of two or more antibodies, referred to in paragraphs (i) to(iv).

In a preferred embodiment of the invention, determining the amount of amyloid beta-peptide is carried out using ELISA.

As used herein, the term "ELISA" means enzyme-linked immunoassay and analysis, in which an unknown amount of a substance-target (amyloid beta peptide) is fixed on the surface, then this surface is applied with a specific antibody to it has been associated with the antigen. This antibody binds to the enzyme and in the final stage adds the substance that the enzyme can convert to some detectibly signal. Various types of ELISA assays known in the art and can be used in the method according to the invention, including direct ELISA, sandwich ELISA, competitive ELISA and a method and apparatus for a reverse ELISA (ELISA-R m&d).

Direct ELISA-analysis carried out by contacting a test sample containing amyloid beta peptide, with solid n�the mixer, which was pre-coated with a concentrated solution of an inert protein or reagent (bovine serum albumin, casein). After absorption of amyloid beta-peptide that is present in the test sample, media, add antibody specific to amyloid beta-peptide under conditions suitable for binding of amyloid beta-peptide. Then bound antibody detected using a second antibody that binds to apparently detected a label or with a substrate-modifying enzyme. The signal produced apparently detected a label or substrate, is proportional to the amount of antibody associated with the carrier, which, in turn, directly correlates with the amount of amyloid beta-peptide in the sample.

Competitive ELISA analysis includes a first stage on which a test sample containing an unknown amount of amyloid beta-peptide is subjected to contacting with the "first" antibody, as defined above. In the hole, covered with the antigen, adding the "antibody-antigen". After washing the medium to remove any non-specifically associated complexes, the number of "first" antibody detects using a "second" antibody associated with apparently detected molecule. In analyses of this type, the higher the original antigen concentration, the weaker the output signal. And�ternational competitive ELISA-test is the ELISA, uses no antibody bound to the enzyme and the antigen associated with the enzyme. The labeled antigen competes with the antigen (unlabeled) sample for binding sites with the "first" antibody. In analyses of this type, the concentration of antigen in the sample is inversely proportional to the amount of labeled antigen remaining in the hole, and accordingly, the greater the concentration, the weaker the signal.

In the method and apparatus for a reverse ELISA (ELISA-R m&d) is applied to the solid phase is a new type made from linked immunosorbent polystyrene rod with 4-12 protruding pointed ends, and the entire device can be put into the test tube containing the collected sample, and subsequent steps (washing, incubation in conjugate and incubation in a chromogenic solution) can be easily carried out by immersion of the pointed ends of the device in microwells in standard microtitre plates pre-filled with reagents and stored in hermetically closed form up to their application.

In a preferred embodiment of the invention, an ELISA-assay is a "sandwich"ELISA. Analysis of the "sandwich"ELISA involves coating the carrier of the "first" antibody specific to amyloid beta peptide, the application of a sample containing amyloid beta-peptide, resulting in the binding of amyloid beta-peptide with�Ervin" antibody and the application of the "second" antibody, which is specific for amyloid beta-peptide-where the "second" antibody is usually associated with apparently detected a label or with a substrate-modifying enzyme. The signal generated by the label or turned by the substrate, is proportional to the amount of antigen in the sample.

In the context of describing the present invention, the "first" antibody is called an "antibody capture", which means that this antibody is used for separation of sample molecules of all kinds, which specifically binds the antibody. The type of antibody that can be used as antibodies for capture, has no particular restrictions, provided that it contains at least one antigen-binding site specific for Αβ40 and/or Αβ42. Thus, as the antibodies for capture can be used any of the above antibodies.

In the context of describing the present invention, the "second" antibody is called a "detection antibody", as this antibody can be used for detection of the amount of antigen that is retained on the antibody for capture. Similarly, the antibody for capture, the type of antibody that can be used as the detecting antibody, has no limits. There are, however, from�etit, what of the detecting antibody (i) shall contact the region of the antigen that has not been sensitized by antibody capture, and (ii) must include the antigen-binding site, but also detectivea label, substrate-modifying enzyme or extra(s) area(s) which(s) may specifically be detected by a reagent that detects high affinity binding with said antibody that will detect the antibody that binds to the antigen captured by the antibody for capture. Preferably, these additional areas that can specifically communicate with the specified reagent, correspond to the constant region of the immunoglobulin molecule.

In a preferred embodiment of the invention, the antibody to capture antibody is specific to N-terminal region of amyloid beta-peptides. In a more preferred embodiment of the invention, the specified antibody to capture antibody is specific for epitope localized in the provisions of amino acids 1-16 Abeta40 or Abeta42. Particularly preferred antibody for capture is a monoclonal antibody 6E10, described Kim, K. S. (Neuroscience Res. Comm. 1988, 2:121-130).

In another preferred embodiment of the invention, the detecting antibody is an antibody specific to the epitope localized in the C-terminal about�Asti amyloid beta-peptide. As explained above, the type of the detecting antibody can be adequately selected by an average person skilled in the art depending on the number of detected amyloid beta-peptides.

For detection or for determining one Αβ40 antibody for capture can serve as an antibody that recognizes the N-terminal region Αβ40 (and Αβ42, since both peptides have identical N-terminal region), and the detecting antibody may be an antibody that specifically recognizes the C-terminal region Αβ40, but shall not enter into any cross-react with Αβ42. Alternative, Αβ40 can be specifically detected using antibodies to capture that recognizes the C-terminal region Αβ40, but shall not enter into any cross-react with Αβ42, and a detection antibody that recognizes the region Αβ40, which is common for Αβ40 and Αβ42, and preferably, the N-terminal region Αβ42/Αβ40.

For detection or for determining one Αβ42 antibody for capture can serve as an antibody that recognizes the N-terminal region Αβ42 (and Αβ42, since both peptides have identical N-terminal region), and the detecting antibody may be an antibody that specifically recognizes the C-terminal region Αβ42, but shall not enter into any cross-react with Αβ40. Alta�natively, Αβ42 can be specifically detected using antibodies to capture that recognizes the C-terminal region Αβ42, and a detection antibody that recognizes the region Αβ42, which is common to Αβ42 and Αβ40.

For simultaneous detection or determination Αβ42 and Αβ40 antibody for capture can serve as an antibody that recognizes the N-terminal region common to Αβ42 and Αβ40 and the detecting antibody may serve as a combination of at least two antibodies, the first antibody specifically recognizes the C-terminal region Αβ42, but shall not enter into any cross-react with Αβ40, and the second antibody specifically recognizes the C-terminal region Αβ40, but shall not enter into any cross-react with Αβ42. Alternatively, the antibody for capture can serve as an antibody that recognizes the N-terminal region common to Αβ42 and Αβ40 and the detecting antibody may be an antibody that recognizes the C-terminal region Αβ40 and Αβ42. Alternative, Αβ40 and Αβ42 can be simultaneously detected by use of antibodies to capture a mixture of at least two antibodies containing the first antibody that specifically recognizes the C-terminal region Αβ42, but shall not enter into any cross-react with Αβ40, and a second antibody that specifically recognizes the C-concave� region Αβ40, but it does not enter into any cross-react with Αβ42, and a detection antibody that recognizes the N-terminal region common to Αβ42 and Αβ40. Alternative, Αβ42 and Αβ40 can be simultaneously detected by using as an antibody to capture antibody that recognizes the C-terminal region Αβ40 and Αβ42, and a detection antibody that recognizes the N-terminal region common to Αβ42 and Αβ40.

Antibodies specific to Αβ40 and Αβ42, and methods for their preparation have been described in detail in the applications WO2004024770 and WO2004098631, the content of which is introduced into the present description by reference.

The detecting antibodies and/or antibody for capture can be affinity purified using a polypeptide containing a polypeptide sequence used to produce these antibodies.

As mentioned above, the detection antibody may be directly connected with apparently detected a label or with a substrate-modifying enzyme. Preferably can be used a reagent having affinity to the detecting antibody, and in this case the specified reagent mark is not detecting the antibody, and apparently detected a label or substrate-modifying enzyme. In addition, the antibody-binding reagent may be associated with the first member of a binding pair, and in this case, he is the second member of tie�equation describing pairs, which may be associated with apparently detected a label or with a substrate-modifying enzyme.

Antibody-binding reagent can be ecovalence is associated with a specific antibody(data) type(s) specific(s) or class(es) and/or specific(governmental) subclass(s) and/or fragments of such antibodies. Alternatively, the antibody-binding reagent can be ecovalence is associated with an antibody specific to a particular antigen. In specific embodiments of the invention, the antibody-binding reagent can be ecovalence is associated with the Fc-region or with F(ab) region of the detecting antibody. Preferred antibody-binding reagents are protein A, G-protein, the V protein, protein L, anti-Fc antibody or antibody-binding fragment and Fc-receptor (FcR) or antibody-binding fragment. Non-limiting examples of antibodies that can ecovalence to contact with the detecting antibody are monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, gumanitarnye antibodies, chimeric antibodies, single-domain antibodies, single-chain Fv (scFv) single chain antibodies, Fab fragments, F(ab') fragments, Fv (sdFv), linked with a disulfide bond, intratel and antiidiotypic (anti - Id) antibodies, and epitope-binding fragments of any of the above antibodies. Non-limiting when�erami Fc-receptor is FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIAα, FcγRIIIB, FcεRIα, FcεRIξ and FcγRIIIAξ.

Suitable binding pairs used for detection are as follows:

- hapten or antigen/antibody, such as digoxin and antibodies against digoxin

- Biotin or Biotin analogues (e.g. iminobiotin, iminobiotin or desthiobiotin)/avidin or streptavidin,

- sugar/lectin,

the enzyme and cofactor,

- folic acid/folate,

- double stranded oligonucleotides that selectively bind to proteins/transcription factors,

- a nucleic acid or nucleic acid analogue/complementary nucleic acid,

receptor/ligand, such as a steroid hormone receptor/steroid hormone.

It should be noted that the term "first" and "second" member of a binding pair is relative, and that each of the above members can be considered as the first or second members of the binding pair. In a preferred embodiment of the invention, the first member of the binding pair is Biotin or a functionally equivalent variant, and the second member of the binding pair is avidin, streptavidin or a functionally equivalent variant.

In a preferred embodiment of the invention, the second member of the binding pair is streptavidin.

Suitable detection labels include, but are not limited to�, fluorescent molecules (e.g. fluorescein, rhodamine, phycoerythrine, coumarin, oxazine Serie, resorufin, cyanine and derivatives thereof), fluorescent molecules (e.g., nanoparticles Qdot™ supplied by the Quantum Dot Corporation, Palo Alto, CA).

Suitable substrate-modifying enzymes are enzymes that have the ability to produce a detected signal, for example, after adding the activator, substrate, amplifier, etc., Enzymes, suitable for use in the present invention as detectable labels, and their respective substrates are:

Alkaline phosphatase:

- Chromogenic substrates: substrates based on p-nitrophenylphosphate (p-NPP), 5-bromo-4-chloro-3-indolylacetate/blue nitrotyrosine (BCIP/NPT), fast red/naphthol-AS-TS-phosphate.

- Fluorogenic substrates: 4-methylumbelliferyl (4-MUP), 2-(5'-chloro-2'-postrelational)-6-chloro-4-(3H)-hintline (CPPCQ), 3,6-fluorescencebased (3,6-FDP), fast blue BB, fast red TR or diazonium salt fast red violet LB.

- Peroxidase:

- Chromogenic substrates based on 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPT), 3,3',5,5'-the tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 3 - dimethylaminobenzoyl acid (DMAB) and 3-methyl-2 - benzothiazolinone (MBTH), tetrahydrochloride 3-amino-9-FL�of carbazole (AEC) and 3,3'-diaminobenzidine (DAB).

- Fluorogenic substrates: 4-hydroxy-3-methoxyphenylalanine acid, restored phenoxazine and restored benzothiazine, including reagent Amplex red®, bright red and Amplex restored dihydroquinine.

- Glycosidase:

- Chromogenic substrate: o-nitrophenyl-β-D-galactoside (o-NPG), p-nitrophenyl-β-D-galactoside and 4-methylumbelliferyl-β-D-galactoside (MUG) for β-D-galactosidase.

- Fluorogenic substrates: β-β D galactopyranoside resorufin, digalactosyl fluorescein (FDG), fluorescein diglucuronide, 4-methylumbelliferyl-β-β D galactopyranoside, carboxymethylcel-β-β D galactopyranoside and fluorinated β-D-galactopyranoside coumarin.

- Oxidoreductase (luciferase):

- Fluorescent substrates: luciferin.

Kits according to the invention

The present invention also relates to kits that are suitable for implementing the method according to the invention. Thus, in another aspect, the present invention relates to a kit for detection of amyloid beta peptides in the biological sample, where the specified set contains:

(i) protein-solubilizers agent and

(ii) at least one antibody against amyloid beta-peptide.

Components (i) and (ii) of this set is essentially described in the method according to the invention. In a preferred embodiment of the invention, baie�OK-solubilizers agent is a detergent. In an even more preferred embodiment of the invention the specified detergent is tween 20.

In another preferred embodiment of the invention, at least one antibody against amyloid beta peptide selected from the group consisting of:

(i) antibodies against N-terminal region of amyloid beta-peptide,

(ii) antibodies against the C-terminal region of amyloid beta-peptide, and

(iii) a combination of antibodies against N-terminal region of amyloid beta-peptide and antibodies against the C-terminal region of amyloid beta-peptide.

In an even more preferred embodiment of the invention, the kit according to the invention contains an antibody against N-terminal region of amyloid beta-peptide, which is directed against an epitope localized in the provisions of amino acids 1-16 ABETA40 and ABETA42. In another preferred embodiment of the invention, the kit according to the invention contains an antibody against the C-terminal region of amyloid beta-peptide, which is directed against the peptide and selected from the group consisting of:

(i) polyclonal antibodies against the peptide corresponding to the C-terminal region of the peptide ABETA42, where the specified antibody specifically binds to ABETA42, but essentially does not enter into any cross-react with ABETA40,

(ii) polyclonal antibodies against the peptide corresponding to the C-terminal region of the peptide ABETA40, where�asanee antibody specifically binds to ABETA40, but, essentially, does not enter into any cross-react with ABETA42, and

(iii) antibodies that recognize the C-terminal region ABETA40 and ABETA42 and

(iv) a combination of antibodies according to paragraphs (i) and (ii).

In a preferred embodiment of the invention, the kit according to the invention also includes solid support. As used herein, the term "carrier" or "surface" means a solid phase, which is water-insoluble, porous or non-porous material that can be manufactured in various forms such as strips, rods, particles, including latex particles, magnetic particles, microparticles, spheres, membranes, microtiter wells microtiter plates and plastic tubing. In principle, as a solid carrier can be any material provided that it will possess the ability to bind to a sufficient number of antibodies for capture. Thus, the choice of solid phase material can be made based on the desired operating parameters of the analysis of this format. Materials suitable for the manufacture of solid media are polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as paper fiber, such as filter paper, chromatographic paper, glass fiber, etc.; synthetic remodification natural polymers, such as nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, cross-linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutane), polystyrene, polymethacrylate, polyethylene terephthalate, nylon, polyvinylbutyrate, etc., which can be used separately or in combination with other materials; glass such as, for example, glass is used as a biological glass, ceramics, metals, etc. are Preferred not associated cross-links the polymers of styrene and carboxylated styrene or styrene with other active functional groups, such as amino, hydroxyl, halogen, etc. In some cases, there may be used copolymers of substituted styrenes and dienes, such as butadiene.

Solid support and the antibody may be supplied in the kit individually, or, alternatively, the carrier may be pre-coated with an antibody to capture. In this case, the carrier may be treated with blocking solution after binding of the antibody to capture.

Additional components of this kit can be:

- Means for taking a patient sample for analysis,

- Buffers and solutions needed to construct standard curves for amyloid beta-peptides,

- Buffers and solutions for washing and lock�of solid media during the analysis,

- Buffers and solutions for the deposition of antibodies on a solid support,

- Reagents for the detection of color or fluorogenic signal produced apparently detected the mark,

- Reagents for the termination of formation of a colored or fluorogenic product produced apparently detected label (for example, 1H H2SO4),

The sample containing the mother liquor peptides Αβ40 or Αβ42 or a combination thereof.

In a preferred embodiment of the invention, the kit according to the invention comprises two antibodies that can be used in a "sandwich"ELISA-analysis. In this case, one of the antibody, i.e., antibody capture, immobilized on a solid support. Immobilization may be performed before binding polypeptide detected is the target or after binding of the peptide/protein with the antibody for capture. In any case, if you are using solid media, it is easier to block excess protein binding sites on the carrier before adding the sample containing the designated polypeptide-target. Preferably, the blocking or neutralization of the peptide-binding sites on the carrier is carried out using the same buffer that was used for washing of complexes after each binding reactions (for example, 50 mm Tris-HCl, pH 8, PBS or TBS containing, but not necessarily, tween 20) and in which was� added macromolecular compound (e.g., bovine serum albumin (BSA, nonfat dry milk blocking reagent used in the Western blot analysis, casein, lacto albumin, ovalbumin) in concentrations of about 0.05% -10%, preferably 1-5%, and more preferably about 3%.

The present invention is described in the following examples, which are illustrative only and should not be construed as limiting the scope of the invention.

Examples

Example 1

Groups participating in the study

The study included 40 participants, including 16 healthy individuals were selected as controls (HC), 8 patients with a weak violation of cognitive abilities, suffering from memory loss (MCI), and 16 patients with Alzheimer's disease (AD). All participants were aged over 65 years, and each group was half of men and women. Demographic characteristics of participants are systematized in table 2.

Table 2
Demographic characteristics
FeaturesBAMCIHCR1
Men/women8/8 4/48/8-
Age (years, mean ± RMS Osh.)78,8±4,7H77,3±3,6H70,3±4,1M,A0,0002
The frequency of occurrence ε4 (%)34,4HA 37.5H3,1M,And0,002
Education*1,9±0,91,6±0,92,4±0,60,072
* Education was assessed according to the following scores: 0 = no education; 1 = primary education; 2 = secondary education; 3 = higher education.1U-criterion of the Kruskal-Wallis contrasts with the U-Mann-Whitney test. H, M, and A mean significant magnitude HC, MCI and BA, respectively.

Healthy individuals were carefully selected from living in the communities socially active volunteers, who had no complaints of memory loss were observed normal behavior, as indicated by neuropsychological tests, and there was no structural change, as indicated by quantitative magnetic resonance �omography (MRI).

Participants with MCI met the clinical criterion Mayo. In addition, for the selection of participants with MCI needs to CDR=0.5, and more than 3 points on a scale of Selten (J. Neurol. Neurosurg Psychiatry. 1992; 55:967-972), indicating atrophy of the medial area of the temporal region at the picture and hypometabolism in the parietal and/or temporal region, defined by positron emission tomography, carried out with 18-Tordesillas (PET-FDG), presumably on BA. Were excluded patients with any mental or systemic pathology, which, presumably, is a neurodegenerative disease, which can cause MCI.

Specific criteria for inclusion in the BA group is a provisional diagnosis of BA (criteria NINCDS-ADRDA), CDR=1, MMSE of 16 of 24 points and score more than 3 points on a scale of Selena for atrophy of medial temporal plot area.

A survey on cognitive abilities for diagnosis for MCI-group and BA group has undertaken in accordance with routine clinical evaluation of memory loss of ACE described in the literature.

From each participant in the studies provided written informed consent (or, in the case of some patients with asthma, the consent of their close relatives). Research protocols were reviewed and approved by the ethics Committee (Ethical Committee of the Hospital Clinic i Povincial (Barcelona, Spain).

Example 2

Obtaining samples

Individuals with blood was taken and added pellets Roche CompleteMini (protease inhibitors), 10 ml each. Blood or centrifuged, or stored at 4°C and centrifuged on the day of analysis.

Plasma was separated from the cell fraction and the plasma was collected, divided into 0.5 ml-aliquots and placed in Eppendorf tubes. Aliquots can be stored at -80°C.

The level of free amyloid in plasma (1ab40 and 1ab42) was determined directly in undiluted clarified plasma, obtained as described below. These parameters are further denoted 1ab40 or UP (undiluted plasma) includes AB(1-40) for these AB(1-40) and 1ab42 or UP includes AB(1-42) Aβ(1-42).

Total plasma amyloid corresponding to the free amyloid in the plasma plus the amyloid associated with components of the plasma were determined in samples obtained by diluting 150 µl of plasma in 300 µl of buffer for dilution (PBS containing 0.5% tween-20). These parameters are further denoted 2ab40 or DP (diluted plasma) includes AB(1-40) for these AB(1-40) and 2ab42 or DP includes AB(1-42) for these AB(1-42).

Associated with cells of amyloid beta determined by dilution of cell fractions of plasma (V/V) 1/5 in the sample for dilution (PBS containing 0.5% tween-20). These parameters are indicated next 3ab40 or CB (associated with cells) includes AB(1-40) for these AB(1-40) and 3ab42 or CB includes AB(1-42) for these AB(1-42).

Example 3

Conditions �of processing sample

The dilutions of the sample

To identify the dilution of the sample with higher optical density, as shown by ELISA analysis, were tested with different dilutions and buffer solutions.

Samples were diluted 1/2, 1/3, 1/4, 1/5 and 1/10. It was found that at a dilution of 1/4 and above, the optical density of the sample decreased. The best dilutions are dilutions of 1/2 and 1/3.

Centrifugation of the sample

Samples can be clarified by centrifugation for 1 minute at 13000 rpm to remove components in the form of large particles, which can have a negative impact on immunological detection. The supernatant can be collected and tested immediately or after dilution as described above.

Processing of the sample by ultrasound

Samples can be treated with ultrasound for 5 to 10 minutes. Ultrasonically treated samples can be used directly in ELISA-tests, or they can be centrifuged for 1 minute at 13000 rpm, and the supernatant can be used directly in ELISA-assays. Ultrasonically treated samples may also be diluted with appropriate buffer as defined in the previous examples.

Pre-bleaching of the sample

The samples were passed through a column of separate 4B-IgGK d�I remove possible impurities, basically as described Fuckumoto et al., (Arch. Neurol, 2003, 60: 958-964). Column separate 4B-IgGK was obtained by reaction between CNBr-activated sepharose with IgGlk in accordance with the following procedure:

- IgGlk (molecular weight (MW)=150000) was dissolved in the buffer for binding (0.1 M NaHCO3, pH 8.3, NaCl, 0.5 M (1.5 ml to 300 mg of agarose)) using 500 ál IgG1k (0.6 mg) and 1.5 ml of the buffer to bind.

- The resin was washed with 1 mm HCl (60 ml for every 300 mg of resin).

The ligand was mixed with the resin, acid washed, and incubated over night at 4°C with constant shaking (alternatively, incubation can be carried out for 2 hours at room temperature).

- Then the excess ligand was washed with 5 volumes of buffer for binding. After that, the unreacted active groups in the resin were blocked using 0.1 M Tris-HCl, pH 8, for 2 hours at room temperature with agitation.

- The resin was washed with in three cycles using, alternatively, 0.1 M Tris-HCl, pH 8, + 0.5 M NaCl/0.1 M sodium acetate, pH 4, +0.5 M NaCl.

After receiving sepharose 4B-IgGK carried out, the following processing stage sample:

To 300 μl of plasma was subjected to contacting with 525 μl of sample buffer and 75 ál of agarose-IgGlk and incubated for 2 hours at 4°C with constant stirring.

- Agarose was removed by centrifugation (5 minutes at 1000 rpm)./p>

- 100 µl of the treated sample were added to wells of microtiter plates, which have been adsorbed antibody for capturing specific to the N-Terminus of amyloid beta-peptides, and incubated over night at 4°C.

- The number of amyloid beta-peptide present in the clarified sample was determined using ELISA analysis by adding to the wells of the anti-Αβ40 or anti-Αβ42 specific antisera (1/4000) for 1 hour at room temperature and under constant shaking.

- Then the samples were incubated with 1/5000-breeding biotinylating anticalcium antibodies for 1 hour at room temperature with constant shaking.

- Then the samples were incubated with 1/4000-breeding streptavidin associated with peroxidase for 1 hour at room temperature, with shaking.

- Then the reaction showed TMB reagent for 30 minutes in the dark.

The reaction was stopped by the addition of the blocking solution, and optical density was read at 450 nm.

The removal of albumin and IgG

The samples were passed through the column, which bind albumin and IgG. The flowing stream was analyzed to identify fractions in the presence of amyloid peptides. Albumin was removed using a set of "ProteoExtract Albumin Removal Kit"(CALBIOCHEM) according to sho�Yami manufacturers.

IgG was removed on a column of protein A (protein a-sepharose 4 Fast Flow, Amersham Biosciences) according to the manufacturers instructions.

The sample concentration

The samples were concentrated on microconcentrators with a cut-off of 10,000. Amyloid peptides were captured in the ongoing flow, and high molecular weight proteins and captured in the UF-retentate.

Effects resulting from treatment in accordance with different protocols, can be systematized as follows:

- Detergents: tween 20 is a detergent that will provide a significant increase in the values of optical density. This effect was not observed if the concentration of tween 20 was increased from 0.05% to 0.1%.

- pH: the pH at which achieved the best results was 7 and 8. When determining Aβ40 adequate optical density was also observed at pH=9. When determining Aβ42 adequate values of optical density were obtained at pH 9 and 5.

- Denaturing conditions: the Addition of 0.5 M or 1 M GuHCl or 10% DMSO did not lead to an increase in optical density.

- Salt: higher values of optical density were obtained using NaCl instead of KCl.

- BSA: Any differences in the levels of optical density was observed in the case of increasing the concentration of BSA with 0.05% to 0.5%.

Processing by ultrasound: Any difference� was not observed in case of samples with ultrasound to determine optical density.

- Preliminary clarification: any effect was not observed in the case of pre-treatment of samples by separate 4B-IgGk.

- Removal of albumin and IgG: Amyloid peptides, obviously, associated with IgG, but not with albumin.

- Concentration: Amyloid peptides are present mainly in the UF-retentate.

Example 4

Colorimetric sandwich ELISA analysis with signal amplification under the action of Biotin-streptavidine

To increase the sensitivity of the signal can be amplified using Biotin-streptavidin. The tablet was covered with antibody (mAb) 6E10 for engagement, which recognizes amino acids 1-17 in the amyloid peptide Aβ40 in amyloid peptide Aβ42. The coating was performed at a concentration of 5 mg/ml in 100 mm carbonate/bicarbonate buffer, pH of 9.6, overnight at 4°C. Then the plate was blocked with 300 μl of a blocking solution (50 mm Tris-HCl, pH 8, 0.2% tween 20 and 0.5% BSA) for 3 hours at room temperature with agitation or for 2 hours at 37°C. If necessary, after blocking, the tablet may be treated with 100 μl of 50 mm solution of Tris-HCl, pH 8, containing 20 mg/ml trehalose. Plates were left to evaporate until then, until it formed a white halo indicating the presence of trehalose. The thus treated plates, wrapped in aluminum foil, can be stored at 4°C isochronal its stability within two years.

Standard curves were constructed by the data obtained for 200 PG/ml stock solution of the peptides Aβ40 and β42 on tablets covered with mAb 6E10 and treated with trehalose. From these solutions were prepared serial dilutions 1:2 in SDB so that the concentration was 200, 100, 50, 25, 12,5, 6,25 and 3.125 PG/ml 100 ál of each diluted or undiluted sample was added to SDB (1/1000000) and incubated over night at 4°C or for 2 hours at 37°C). Samples for determination of free plasma amyloid, total plasma amyloid and amyloid associated with the cells in the test samples were prepared as described in example 1 and added to the wells of the ELISA-plates in conditions similar to the conditions of preparation of the standard sample. Then added the detecting antibody (polyclonal antibody against the peptide corresponding to the C-terminal region of the peptide Αβ42 or polyclonal antibody against the peptide corresponding to the C-terminal region of Αβ40 peptide, depending on specifically detected peptide, i.e., Αβ42 Αβ40 or) diluted in SDB. To each well was added 100 µl of antibody and incubated for 1 hour at room temperature. After this was added 100 µl of Biotin-labeled antibodies against rabbit IgG (SIGMA) at a dilution of 1/5000 in SDB and incubated for 1 hour at room temperature with agitation. Then in CA�blow hole was added 100 μl of HRP-conjugated of streptavidin (from SIGMA) at a dilution of 1/4000 in SDB and incubated for 1 hour at room temperature.

The tablet was shown using 100 ál of chromogenic substrate TMB (ZEU Inmunotec). To do this, TMB was added and incubated in the dark for 15-30 minutes. Then to each well was added 50 μl 1N H2SO4used as a blocking solution. The optical density at 450 nm was read on a tablet reader Synergy HT (BioTek Instruments).

Concentrations (PG/ml) obtained for samples used to determine total plasma amyloid (free amyloid plasma together with the amyloid associated with components of the plasma), corrected for an amendment to the breeding carried out during the stage of preparation of the sample. Since breeding was typically 1:3 (see example 1), the value PG/ml obtained after the read optical density was multiplied by 3 to determine the actual concentration of units free of amyloid in the plasma and amyloid associated with components of the plasma. Similarly, the value PG/ml, obtained from the values of optical density for the samples used to determine the associated with cells amyloid, also corrected for an amendment to the breeding carried out during the stage of preparation of the sample. Since breeding was typically 1:5 (see example 1), the value PG/ml, obtained after reading the optical density of slip casting�and, was multiplied by 5 to determine the actual concentration of amyloid protein associated with the cell.

In between stages the tablet were washed in the automatic device for washing tablets (Elx50 Bio Tek Instruments) programmed for 5 palasciano through each period of time. The flushing solution contained 50 mm Tris-HCl, pH 8, and 0.05% tween 20 and 150 mm NaCl (filtered before use).

Example 5

Fluorescent "sandwich"ELISA-analysis

The tablet was covered with antibody 6E10 in bicarbonate buffer (5 μg/ml) overnight at 4°C. Then the plate was blocked for 3 hours at room temperature with shaking (300 ál/well). Then the test samples and the samples to construct a standard curve was added to the plates and incubated over night at 4°C. To each well was added 1/4000-dilution of detection antibody (anti-Aβ40 or anti-Aβ42 antisera) and incubated for 1 hour at room temperature with agitation. After that I added a serial dilution of FITZ-conjugated antibody (dilution 1/1000, 1/5000, 1/10000) and incubated for 1 hour at room temperature in the dark. The fluorescence was read at an excitation wavelength of 485 nm and emission wavelength of 528 nm.

Alternatively, the analysis was performed using a fluorescent substrateQuanta-Blu(PIERCE), which increases h�stateliest ELISA analysis. Maximum excitation was observed at 325 nm and the maximum emission was observed at 420 nm. This excitation can be detected in the wavelength range of 315-340 nm, and the radiation can be detected in the wavelength range 370-470 nm. QuantaBlu working solution was prepared by mixing 9 parts of a solution of substrate QuantaBlu with one part stable peroxidase solution QuantaBlu (solution is stable for 24 hours at room temperature). Incubation can be carried out during the period of time from 1.5 minutes to 90 minutes at room temperature, and the readout can be carried out after completion of the reaction or in the reaction process without completion (receive blue color).

The tablet was covered with antibody (mAb) 6E10 in bicarbonate buffer (5 μg/ml) overnight at 4°C and then blocked for 3 hours at room temperature with shaking (300 ál/well). Then received different standard curves using the following concentrations of peptide Αβ42 and Αβ40:

- 1000, 500, 250, 125, 62,5, 31, 25 and 15,65 PG/ml

- 200, 100, 50, 25, 12,5, 6,25 and 3.125 PG/ml

- 25, 12,5, 6,25, 3,125, 1,56, 0,78 and 0.39 PG/ml

- 10, 5, 2,5, 1,25, 0,625, 0,3125 and 0,156 PG/ml

- 5, 2,5, 1,25, 0,625, 0,3125, 0,156 and 0,078 PG/ml

- 1, 0,5, 0,25, 0,125, 0,0625, 0,03125 and 0,0156 PG/ml.

Detection antibody (anti-Aβ40 or anti-Aβ42 the antiserum) was added (at a dilution 1/4000) in 1 hour at room temperature with agitation. Then add HRP-konjugierten antibody against rabbit IgG at a dilution of 1/1000 and incubated for 1 hour at room temperature with agitation. For the manifestation of color of the reaction mixture were added 100 µl of the working solution Quanta-Blue and incubated for 30 minutes, 60 minutes and 90 minutes at room temperature in the dark. Then read fluorescence (excitation: 360/40 nm; emission: 460/40 nm) for 30 minutes, 60 minutes and 90 minutes, without completing the reaction, or after completion of the reaction with blocking solution.

Example 6

The construction of the standard curves according to the data for Αβ40 and Αβ42

In order to build the standard curve for Αβ40, lyophilized sample of human β40 was diluted to 10 μg/ml Of stock solution was obtained, the samples containing the following concentrations (in PG/ml): 25000 PG/ml, 2500 PG/ml, 25 PG/ml and 12.5 PG/ml to 6.25 PG/ml of 3.125 PG/ml to 1.56 ng/ml to 0.78 ng/ml. Samples were prepared in the presence of 1 mm of the protease inhibitor AEBSF. Then the samples were processed by the method described in the previous examples.

In order to build the standard curve for Αβ42, lyophilized sample β42 person were diluted to 10 μg/ml Of stock solution was obtained, the samples containing the following concentrations (in PG/ml): 25000 PG/ml, 2500 PG/ml, 25 PG/ml and 12.5 PG/ml to 6.25 PG/ml of 3.125 PG/ml to 1.56 ng/ml to 0.78 ng/ml. Samples were prepared in the presence of 1 mm inhibit�RA protease AEBSF. Then the samples were processed by the method described in the previous examples.

Example 7

Statistical analysis

The reproducibility of measurements in different laboratories were evaluated for 16 randomly selected samples on the correlation coefficient and the concordance (CCC), which allowed to assess the conformity between the three readings, that is, read by the author in his laboratory, and reading of the same sample carried out in two different laboratories by measuring the deflection of 45° from a line passing through the origin (lines of concordance)27. The degree of similarity between samples obtained from the same individual on different days (reproducibility for the same individual), was assessed by the coefficient of intra-group correlation (ICC). The degree of similarity, measured by the correlation coefficients was set to low (from 0.21 to 0.40), moderate (0,41-0,60), high (0,61-0,80) and almost absolute (0,81-1,00). The levels of these AB for different diagnostic groups were compared using the U-Mann-Whitney test. To assess correlations between continuous variables used the analysis of Spearman. To reject the null hypothesis it is necessary that p<0,05. All statistical analyses, including the assessment of diagnostic accuracy, was carried out using a computer program AS 9.1. The graphs shown in Fig. 1-3, constructed using the statistical computer program PASW.

The following indices of reliability and validity of markers of amyloid beta-peptides were evaluated in a screening test for patients suffering from ad, MCI and HC.

For each designated and specific marker analyzed, specifically described below, and the participants were divided into groups: healthy individuals (control) - patients with asthma; healthy individuals (control) - patients with MCI and patients with MCI BA:

Table 3
Classification of patients into groups: patients with asthma and healthy individuals (control) (TP: true positive; FP: false positive; TN: true negative; FN: false negative)
Real classification of participants
BAGear
Classification of participants conducted using markersBATRFP
GearFNTN

Table 4
Classification of patients into groups: patients with MCI and healthy individuals (control) (TP: true positive; FP: false positive; TN: true negative; FN: false negative)
Real classification of participants
MCIGear
Classification of participants conducted using markersMCITRFP
GearFNTN

Table 5
Classification of patients into groups: patients with asthma and patients with MCI (TP: true positive; FP: false positive; TN: true negative; FN: false negative)
Real classification of participants
BAMCI
Classification of participants conducted using markers BATRFP
MCIFNTN

Validation criteria:

Sensitivity:means the probability of correct classification of patients of individuals, i.e., the probability of obtaining a positive result for the individual patient in this test. Therefore, the sensitivity refers to the ability of this test to diagnose the disease.

TP
-----
Sensitivity=
TP+FN

Specificity: means the probability of correct classification of healthy individuals, i.e., the probability of obtaining a negative result for a healthy individual. In other words, specificity can be defined as the ability to identify a healthy individual.

TN
-----
Specificity=
TN + FP

Reliability test:

The positive predictive value: means the probability of having a disease if the test gives a positive result. Therefore, the predicted value may be determined based on the number of patients with a positive test result, which, ultimately, was sick with this disease:

TN
-----
PPV=
TN + FP

The negative predictive value is: means the probability that an individual with a negative test result is actually healthy. This value can be determined by dividing the number of true negatives by the total number of patients with a negative test result:

TN
-----
NPV=
TN + FN

In addition to concepts such as sensitivity, specificity and predictive values, can be considered also such thing as a likelihood ratio, the ratio of probabilities or odds ratio. The last parameter means more likely a specific (positive or negative) result from the viewpoint of the presence or absence of the disease.

Positive likelihood ratio positive or odds ratio: this ratio is calculated by dividing the probability of a positive result in patients patients on the probability of a positive result in healthy individuals. In short, this ratio represents the ratio of the number of true positive results (sensitivity) to the number of false positives (1-specificity):

PLR=HIinCtinandtelbnoCtb1CpeCandfandhnoCtb

Negative likelihood ratio or negative odds ratio: this ratio is calculated by dividing the probability of a negative result regarding the presence of the disease on the likelihood of a negative result regarding the absence of the disease. Therefore, this ratio is calculated as the ratio of the number logroot�negative results (1-sensitivity) to the number of true negatives (specificity):

NLR=1HIinCtinandtelbnoCtbCpeCandfandhnoCtb

ROC curve is presented in the form of a graph and the area under the ROC curve with 95% CI, real classification in accordance with the parameter (sick/healthy) patients, the values of sensitivity, magnitude, specificity, positive predictive value and negative predictive value with 95% CI are presented in tables.

All statistical tests were conducted with a significance level of 0.05.

Example 8

The reproducibility of the measurement results of these AB obtained in different laboratories

Sixteen randomly selected samples taken from any party, and their extracts were sent to two independent laboratories to assess reproducibility of measurements obtained in different laboratories. All markers have acted similarly in accordance with the CCC, which ranged from 0.84-0.99 (no total 95% IC ranged from 0.73 to 0.99), which corresponds, in all cases, the degree of similarity is assessed as a high degree of similarity or nearly so direct�deleterious similarity (Fig. 1).

Average vnutrennepolytycheskoy reproducibility of measurement results for six markers conducted in each laboratory, expressed as the coefficient of variability for holes with three replications, was 4,31, of 5.83 and of 8.34 (table 6). Detection limit of the analyses conducted in three laboratories, at 5.31, 3,63 and 1.91 ng/ml for Αβ1-40 and 2.37, and 2,04 2,45 PG/ml for Αβ1-42.

Example 9

Inside individual reproducibility of measurements includes AB

The reproducibility of the measurement results of these AB with four weekly blood samplings (BS1-BS4), measured by ICC, ranged from high to almost absolute for all direct markers in the three groups (table 7). On average, for the three diagnostic groups, a higher ICC consistent with the results of measurements of Αβ1-40 and Αβ1-42 in DP (with 0.93, 95% CI = 0.98 to to 0.80 and 0.93, 95% CI = 0.98 to 0,78; respectively).

Example 10

Comparison of diagnostic groups

In accordance with the highest additionreproducibility of measurement results comparison between groups of participants gave the same pattern for the four blood samples taken on different days (BS1-BS4), although some p-values for these blood samples (BS) varied very slightly. The following description is given based on the measured�tions BS4.

The first unexpected result was that the concentration of Αβ1-40 and Αβ1-42, measured in undiluted plasma (UP), was approximately only 1/3 and 1/4, respectively, from the levels in diluted plasma (DP) for all diagnostic groups (table 8).

Secondly, it was found that levels of the peptide associated with the cells (CB), directly measured in the cellular fraction of the blood samples were similar to levels measured in DP. In addition, the levels of Αβ1-40 and Αβ1-42 clearly correlated with the levels measured in UP, DP or CB-fraction (r = 0,58, 0,71 and of 0.71, respectively; p<0,001). Significant correlations were also found between any pair of the six markers that are directly analyzed in the samples (Αβ1-40 and Aβ1-42 in UP, DP, CB; ( table 9)).

In addition, the authors present invention it was found that in patients with MCI and BA levels of each marker are higher than those of healthy individuals control group (Fig.2, table 8). This increase reached statistical significance in the group of patients with MCI and HC groups for the three markers Αβ1-40 (UP, DP and CB, the levels of which increased 2.9, 2.2, and 1.8, respectively) and for two markers Αβ1-42 plasma (UP and DP, the levels of which were increased by 3.1 and 1.8, respectively). The average level of each marker in the group with asthma was almost analogues�Yong its average level in the group with MCI, and any significant differences between the two groups of patients were observed. Similarly, no statistical differences between the group of patients with asthma and a group of healthy control patients were observed, except for the levels of CB-Αβ1-42 (Fig. 2). This lack of significance, in all probability, caused by a wide range of individual measurements within the group with asthma (n=16), giving the coefficients of variability (CV) of 1.5-2.7 times more than in the group with MCI (n=8), and 2.5 to 5.7 times more than in the HC group (n=16), for each marker (table 8).

Markers of plasma Αβ1-40 and Αβ1-42 (UP and DP), but not CB, correlated significantly with MMSE (table 9), although the level may be somewhat overstated, since the clustering HC tends to increase punctuation. Indeed, with the exception of participants with MMSE ≥ 26, the levels of Αβ1-40 and Αβ1-42 in some patients with severe pathologies (MMSE ≤ 21, n=5) were lower than in patients with moderate disease (MMSE 22-25, n=12), although this difference did not reach statistical significance (data not shown). In addition, three markers Αβ1-42, but not Αβ1-40, found significant correlation with the degree of atrophy of the medial part of the temporal area in the right and left hemisphere (table 9).

The authors of the present invention was also examined multiple markers to be calculated from the markers directly proanalizirovat�tions in the samples. In addition to the regular relations Αβ1-42/Αβ1-40, the most interest is the amount of DP+CB-Αβ1-40 and the sum of DP+CB-Αβ1-42, which were designated by the authors as common Αβ1-40 (T40) and General Αβ1-42 (T42), respectively, and the sum of these two quantities, which have been identified by the authors as common βΑΡΒ (Τ-βΑΡΒ). Relationship Αβ1-42/Αβ1-40, measured for UP, DP or CB, they did not find any significant differences between the groups. However, in the MCI group, the levels T40, T42 and T-βΑΡΒ grew at 2.0, 1.5 and 1.8 times in comparison with levels in the control group of healthy individuals (p≤0,03) (table 4). Similar to the average increase in these levels was observed in healthy control individuals (HC) and patients with BA, but in this case, statistical significance was only given T42 (Fig. 2).

Example 11

Diagnostic features of direct and calculated markers

Direct and derived parameters mentioned in table 10, were determined by methods described in the previous examples.

Table 10
A list of direct and calculated parameters that were analyzed during the study
Direct
Αβ40Αβ42
1ab40 (UP)1ab42 (UP)
2ab40 (DP)2ab42 (DP)
3ab40 (CB)3ab42 (CB)
Calculated parameters
The amount of markers Αβ40The amount of markers Αβ42
1ab40+2ab401ab42+2ab42
1ab40+3ab401ab42+3ab42
2ab40+3ab40/T402ab42+3ab42/T42
1ab40+2ab40+3ab401ab42+2ab42+3ab42
The amount of markers Αβ40 and Αβ42
1ab40+2ab40+1ab42+2ab42
1ab40+3ab40+1ab42+3ab42
2ab40+3ab40+2ab42+3ab42/T-βAPB
1ab40+2ab40+3ab40+1ab42+2ab42+3ab42

Prognostic value of the above parameters were tested for:

(i) diagnosis of Alzheimer's disease (by comparing samples from healthy individuals, with samples taken from patients with Alzheimer's, BA/HC),

(ii) diagnosis of weak cognitive abilities and stage of predecessor�setup portion of Alzheimer's disease (by comparing samples taken from healthy individuals, with samples taken from patients suffering from mild disruption of cognitive abilities, MCI/HC), and

(iii) weak differentiation of cognitive abilities from Alzheimer's disease (by comparing samples taken from patients suffering from mild disruption of cognitive abilities, with samples taken from patients with Alzheimer's disease, MCI/BA).

The diagnostic properties of these tests was evaluated by logistic analysis of the measurement results of these AB and clinical diagnosis, considered as the "gold standard". The results concerning the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy and area under the curve of the working characteristics of the receiver (ROC curve), systematized in table 11.

The most direct markers and two computed marker (T40 and Τ-βΑΡΒ) satisfy the criteria considered appropriate to differentiate patients with MCI from healthy control individuals (ZK), and these markers are of the greatest interest from any practical point of view, especially when it is necessary to establish a more precise diagnosis. Thus, all direct markers, with the exception of CB Αβ1-42, pre�provided on the ROC curve ≥ 0,8, and four of these markers (DP Αβ1-40, CB Αβ1-40, UP Αβ1-42 and DP Αβ1-42) give an accuracy of >80%, meaning that 80% of all test results are correct compared to the clinical gold standard (Fig. 3). The calculated markers T40 and Τ-βΑΡΒ give the same accuracy in the case of differentiation of the group with MCI from HC group, in contrast to the direct marker, and marker T42 gave less reliable results (Fig. 3). Due to the high variability of the measurement results of these AB between individuals in the group with BA was not detected in any of the critical point at which these markers could differentiate patients with BA from individuals of the other two groups with acceptable sensitivity and specificity (Fig. 3).

Example 12

Other parameters indicating the highest sensitivity and specificity, and suitable for use in the present invention, parameters are presented in table 12.

Have been developed that are particularly suitable for differentiating individuals with a weak disruption of cognitive ability from healthy individuals, and in which the marker 1ab40 (figure 4), the marker 2ab40 (figure 5), marker 3ab40 (Figure 6), a marker 1ab42 (figure 7), the marker 2ab42 (figure 8), the marker 3ab42 (figures 9 and 10), marker 2ab40+3ab40 (figure 11), marker 2ab42+3ab42 (figure 12), 2ab42+3ab42(figure 13) and 2ab40+3ab40+2ab42+3ab42 (figure 14).

1. A method of diagnosing a neurodegenerative disease in an individual, comprising the following stages:
(i) determining one or more parameters selected from the group consisting of:
(a) 3ab40 or
(b) the values of the calculated parameter selected from the group consisting of 2ab40+3ab40, 2ab40+3ab40+2ab42+3ab42 and 1ab40+2ab40+1ab42+2ab42;
(ii) comparing the value of the parameter (a) or the amount computed parameter (b) with a reference value corresponding to the value of the specified parameter (a) calculated or specified parameter (b) in the reference model,
where the reference value used in stage (ii) to compare the value of the parameter or the values of the calculated parameter selected from the group consisting of:
(i) to 71.9 PG/ml for parameter 3ab40,
(ii) 132,7 PG/ml for parameter 2ab40+3ab40,
(iii) 235,5 PG/ml for parameter 2ab40+3ab40+2ab42+3ab42,
(iv) 158,3 PG/ml for parameter 1ab40+2ab40+1ab42+2ab42; and
(iii) diagnosis of neurodegenerative diseases, in case there is an increase in the parameter value or values of the calculated parameter is compared with a reference value,
where the specified biological sample is plasma or blood,
where the neurodegenerative disease is Alzheimer's disease and
where
1ab40 corresponds to the level of the free peptide ABETA in a biological sample of the individual;
1ab42 corresponds to the level of free p�of ptid AVITA in a biological sample of the individual;
2ab40 corresponds to the level of free peptide aggregates AVITA in a biological sample of the individual and peptide ABETA associated with the components of the specified biological sample, where 2ab40 determined by calculating the number of peptide ABETA in a specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the peptide ABETA of the components present in the biological sample;
2ab42 corresponds to the level of free peptide aggregates AVITA in a biological sample of the individual and peptide ABETA associated with the components of the specified biological sample, where 2ab42 determined by calculating the number of peptide ABETA in a specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the peptide ABETA of the components present in the biological sample;
3ab40 corresponds to the level of peptide ABETA associated with cells in a biological sample of the individual, where 3ab40 determined by calculating the number of peptide ABETA after contacting the cellular fraction of the specified biological sample with a protein-solubilizers agent under conditions suitable for the stimulation of dissociati� amyloid beta-peptides from the cells, present in a given sample; and
3ab42 corresponds to the level of peptide ABETA associated with cells in a biological sample of the individual, where 3ab42 determined by calculating the number of peptide ABETA after contacting the cellular fraction of the specified biological sample with a protein-solubilizers agent under conditions suitable for the stimulation of dissociation of amyloid beta-peptides from cells present in a given sample.

2. Method of detection of a stage prior to a neurodegenerative disease, where the method comprises the steps:
(i) determining one or more parameters selected from the group consisting of:
(a) 3ab40 or
(b) the values of the calculated parameter selected from the group consisting of 2ab40+3ab40, 2ab40+3ab40+2ab42+3ab42 and 1ab40+2ab40+lab42+2ab42;
(ii) comparing the value of the parameter (a) or the amount computed parameter (b) with a reference value corresponding to the value of the specified parameter (a) calculated or specified parameter (b) in the reference model,
where the reference value used in stage (ii) to compare the value of the parameter or the values of the calculated parameter selected from the group consisting of:
(i) a 71.1 PG/ml for parameter 3ab40,
(ii) 132,7 PG/ml for parameter 2ab40+3ab40,
(iii) 235,5 PG/ml for parameter 2ab40+3ab40+2ab42+3ab42,
(iv) 's 142.4 ng/ml for parameter 1ab40+2ab40+lab42+2ab4;
and
(iii) detecting the disease stage prior to a neurodegenerative disease, in case there is an increase in the parameter value or values of the calculated parameter is compared with a reference value,
where the specified biological sample is plasma or blood,
where stage prior to a neurodegenerative disease, is a weak violation of cognitive abilities, and
where
1ab40 corresponds to the level of the free peptide ABETA in a biological sample of the individual;
1ab42 corresponds to the level of the free peptide ABETA in a biological sample of the individual;
2ab40 corresponds to the level of free peptide aggregates AVITA in a biological sample of the individual and peptide ABETA associated with the components of the specified biological sample, where 2ab40 determined by calculating the number of peptide ABETA in a specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the peptide ABETA of the components present in the biological sample;
2ab42 corresponds to the level of free peptide aggregates AVITA in a biological sample of the individual and peptide ABETA associated with the components specified biological �of brazza, where 2ab42 determined by calculating the number of peptide ABETA in a specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the peptide ABETA of the components present in the biological sample;
3ab40 corresponds to the level of peptide ABETA associated with cells in a biological sample of the individual, where 3ab40 determined by calculating the number of peptide ABETA after contacting the cellular fraction of the specified biological sample with a protein-solubilizers agent under conditions suitable for the stimulation of dissociation of amyloid beta-peptides from cells present in a given sample; and
3ab42 corresponds to the level of peptide ABETA associated with cells in a biological sample of the individual, where 3ab42 determined by calculating the number of peptide ABETA after contacting the cellular fraction of the specified biological sample with a protein-solubilizers agent under conditions suitable for the stimulation of dissociation of amyloid beta-peptides from cells present in a given sample.

3. Method of differentiation of neurodegenerative disease from a stage preceding the specified neurodegenerative disease, where the method comprises the steps:
(i) determine�Oia one or more parameters, selected from the group consisting of:
(a) 3ab40 or
(b) the values of the calculated parameter selected from the group consisting of 2ab40+3ab40, 2ab40+3ab40+2ab42+3ab42 and 1ab40+2ab40+1ab42+2ab42;
(ii) comparing the value of the parameter (a) or the amount computed parameter (b) with a reference value corresponding to the value of the specified parameter (a) calculated or specified parameter (b) in the reference model,
where the reference value used in stage (ii) to compare the value of the parameter or the values of the calculated parameter selected from the group consisting of:
(i) to 211.3 PG/ml for parameter 3ab40,
(ii) 550,8 PG/ml for parameter 2ab40+3ab40,
(iii) 778,1 PG/ml for parameter 2ab40+3ab40+2ab42+3ab42,
(iv) 161,2 PG/ml for parameter 1ab40+2ab40+lab42+2ab42;
and
(iii) differentiation of neurodegenerative disease from a stage preceding the specified neurodegenerative disease, if there is an increase in the parameter value or values of the calculated parameter is compared with a reference value,
where the specified biological sample is plasma or blood,
where the neurodegenerative disease is Alzheimer's disease, and stage prior to a neurodegenerative disease, is a weak violation of cognitive abilities, and
where
1ab40 corresponds to the level of the free peptide ABETA in a biological sample of the of individuals is big�a;
1ab42 corresponds to the level of the free peptide ABETA in a biological sample of the individual;
2ab40 corresponds to the level of free peptide aggregates AVITA in a biological sample of the individual and peptide ABETA associated with the components of the specified biological sample, where 2ab40 determined by calculating the number of peptide ABETA in a specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the peptide ABETA of the components present in the biological sample;
2ab42 corresponds to the level of free peptide aggregates AVITA in a biological sample of the individual and peptide ABETA associated with the components of the specified biological sample, where 2ab42 determined by calculating the number of peptide ABETA in a specified sample after the contacting of the specified sample with protein-solubilizers agent under conditions suitable for the stimulation of dissociation of the peptide ABETA of the components present in the biological sample;
3ab40 corresponds to the level of peptide ABETA associated with cells in a biological sample of the individual, where 3ab40 determined by calculating the number of peptide ABETA after contacting the cellular fraction of the specified bio�oricheskogo sample with protein-solubilizers agent under conditions suitable to stimulate the dissociation of amyloid beta-peptides from cells present in a given sample; and
3ab42 corresponds to the level of peptide ABETA associated with cells in a biological sample of the individual, where 3ab42 determined by calculating the number of peptide ABETA after contacting the cellular fraction of the specified biological sample with a protein-solubilizers agent under conditions suitable for the stimulation of dissociation of amyloid beta-peptides from cells present in a given sample.

4. A method according to any one of claims. 1-3, where the specified protein-solubilizers agent is a detergent.

5. A method according to any one of claims. 1-3, where the step of identifying at least one or more 1ab40, 1ab42, 2ab40, 2ab42, 3ab40 and 3ab42 is carried out by immunoassay.

6. A method according to claim 5, where the specified by immunoassay is an ELISA-analysis.

7. A method according to claim 6, where the specified ELISA-analysis is a "sandwich"ELISA-analysis.

8. A method according to claim 7, where the antibody to capture in the "sandwich"ELISA-analysis is an antibody against N-terminal region of amyloid beta-peptide.

9. A method according to claim 8, wherein the antibody against the N-terminal region of amyloid beta peptide directed against an epitope localized in the provisions of amino acids 1-7 AVETA and AVATA.

10. A method according to any one of claims. 6-9, where detecting an�iteam in the "sandwich"ELISA-analysis is an antibody, specific to the epitope, localized in the C-terminal region of amyloid beta-peptide.

11. A method according to claim 10, wherein the antibody specific to C-terminal region of amyloid beta peptide selected from the group consisting of:
(i) polyclonal antibodies against the peptide corresponding to the C-terminal region of the peptide ABETA, where the specified antibody specifically binds to ABETA, but essentially does not enter into any cross-react with AVETA,
(ii) polyclonal antibodies against the peptide corresponding to the C-terminal region of the peptide ABETA, where the specified antibody specifically binds to ABETA, but essentially does not enter into any cross-react with AVETA,
(iii) antibodies that recognises simultaneously the C-terminal region AVETA and ABETA, and
(iv) combinations of antibodies (i) and (ii).

12. A method according to any one of claims. 7-9, where the specified detection antibody is also detected using a reagent having affinity to a specific antibody that binds to the first member of the binding pair.

13. A method according to claim 12, wherein the detection is carried out using the second member of the binding pair associated with apparently detected the tag.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to a method for the prediction of acquired myopia in school children. The substance of the method consists in measuring blood haemoglobin concentrations in 6-8-year-old school children to detect haemoglobin deficiency as shown by the difference of an optimum haemoglobin concentration specific for the above age and an actual haemoglobin concentration in a child. If observing no haemoglobin deficiency at the age of 6-8 years old, a low risk of acquired myopia is predicted. The haemoglobin deficiency to 1.7 g/l enables predicting a risk of acquired low myopia. If the haemoglobin deficiency is 1.7 g/l and more, a high risk of progressive myopia to be developed into moderate or high myopia is predicted.

EFFECT: using the declared method enables developing the reliable and accessible method for the prediction of myopia in the 6-8-year-old children.

2 tbl, 1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to method of diagnosing rheumatoid arthritis, method of determining therapeutic agent for treatment of rheumatoid arthritis and set for realisation of methods. Methods are characterised by the fact that include stage of measuring amount of talin in plasma or serum of animal subject. Said measurement is carried out, for instance, by immunologic method with application of antibody, binding with talin. If amount of talin is higher than its average value in control subject without rheumatoid arthritis, rheumatoid arthritis is diagnosed in subject. In case of reduction of talin amount after introduction of therapeutic agent in comparison with amount of talin before introduction, therapeutic effect is stated. Set in accordance with claimed invention contains solid-phase carrier, to which antibody, binding with talin, is attached.

EFFECT: increased efficiency of diagnostics.

9 cl, 4 tbl, 4 ex, 3 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a method of predicting a probability of reduction of the glomerular filtration rate (GFR) after 3 months of observation after aortocoronary bypass surgery without artificial blood circulation (ACBS without ABC). The essence of the method consists in the fact that the concentration of a kidney injury molecule of type 1 (KIM-1) is determined in blood serum, the ratio of the biomarker KIM-1 concentrations in two time points after 48 hours and 7 days after the operation is calculated and if its value is higher than 1.5, a conclusion about the probability of (GFR) reduction in the remote period after ACBS without ABC is made.

EFFECT: application of the claimed method makes it possible to predict the probability of the glomerular filtration rate (GFR) reduction after 3 months of observation after aortocoronary bypass surgery without artificial blood circulation in an efficient and accurate way.

1 tbl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: early diagnostic technique for pulmonary thromboembolism consists in the fact that if the patient shows any clinical signs indicative of the probability of pulmonary thromboembolism, blood serum fibrin D-dimer is supposed to be measured; if the fibrin D-dimer is less than 0.5 mg/l, a suspected case of pulmonary thromboembolism is cleared of, whereat blood serum fibrin D-dimer of 0.5 mg/l and more requires measuring blood serum interleukin-8 additionally by enzyme immunoassay, and if the measured concentration is 21.3 pg/ml and more, pulmonary thromboembolism is diagnosed.

EFFECT: more accurate and informative diagnostics.

2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology, namely to obtaining oligopeptide compounds, containing a motive, interacting with a proliferating cell nuclear antigen (PCNA) and can be used in medicine. The oligopeptide compound consists of 14-70 amino acids and contains. a PCNA-interacting motive, representing [K/R]-[F/Y/W]-[L/I/V/A]-[L/I/V/A]-[K/R], at least one signal sequence of nuclear localisation and at least one signal sequence of penetration into a cell, with the PCNA-interacting motive being located towards an N-end relative to the signal sequence.

EFFECT: invention makes it possible to carry out the efficient treatment of hyperproliferative disorders by the application of the oligopeptide compound in cyctostatic therapy or in radiotherapy as a sensitising substance.

34 cl, 6 dwg, 4 tbl, 8 ex

FIELD: biotechnology.

SUBSTANCE: on paramagnetic particles bearing the immobilised bacterial protein G of the family Streptococcus, with the blocked solution Denhardt-DNA the protein BoNT/A is adsorbed using specific highly affinity polyclonal antibodies. The formation of the protein complex BoNT/A is detected with a biotin-conjugated antibody by a noncovalent conjugate of DNA fragments with neutravidin. PCR amplification of the DNA matrix is carried out with the fluorescence detection of the signal in real time. Registration of the presence of BoNT/A in the samples under study is carried out by the change in the level of fluorescence compared to the control ones.

EFFECT: effective method of determining the presence of the compound.

5 dwg, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, namely to a method of predicting the development of severe sepsis. The essence of the method consists in the following: on the first day after acute poisoning with substances of narcotic action the content of procalcitonin, interleukin-6 and interleukin-8 is determined in blood serum. If the value of procalcitonin is from 0.5 ng/ml to 5 ng/ml, interleukin-6 from 20 pg/ml to 150 pg/ml, interleukin-8 from 50 pg/ml to 300 pg/ml, the development of severe sepsis is predicted.

EFFECT: application of the claimed method ensures the high reliability of prediction of severe sepsis development.

1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of molecular genetics, genosystematics and pharmacognosy and is intended for the identification of the species affinity of common heather (Calluna vulgaris (L.) Hull.). Claimed is a set of synthetic oligonucleotides for PCR with the fragment ITS2 of nuclear DNA, including forward and reverse primers and a degradable probe.

EFFECT: set possesses high sensitivity and specificity and makes it possible to carry out the identification of a medicinal plant in a fast and reliable way.

1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention aims at detecting benign and malignant new growths in human thyroid. Involved thyroid and reference adjacent intact tissues are sampled; micro-RNA is recovered from the samples; that is followed by conducting a reverse transcription reaction, measuring an expression level of microRNA-21, -221, -222, -155, -205 by real-time RNA followed by a comparative analysis of the microRNA expression according to the norm and thyroid tumour involvement, and stating the presence and type of the new growth. If the above microRNA expression varies by no more than 4 times to the higher and lower figures of expression in relation to the reference, the benign new growth is stated. The malignant new growth is shown by the measured microRNA expression by more than 4 times.

EFFECT: effective detection of the benign and malignant thyroid new growths that promotes improving the further therapeutic approach.

5 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: present group of inventions refers to medicine, namely to cardiology, and concerns diagnosing various forms of the left ventricular hypertrophy. That is ensured by determining an amount of a necrosis marker - cardiac troponin T, cardiac function marker - NT-proBNP and one of an inflammation marker - GDF-15. Comparing their amounts to the reference values enables diagnosing a physiological or pathological left ventricular hypertrophy. Besides, the presence of the pathological hypertrophy requires determining the relation between the cardiac function marker and inflammatory marker, as well as between the necrosis marker and inflammatory marker, and comparing these relations to the reference enables stating if the individual suffers from hypertrophic nonobstructive cardiomyopathy, or hypertrophic obstructive cardiomyopathy, or hypertrophy accompanying pressure overload.

EFFECT: improving the diagnostic procedure.

7 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: patient's synovial fluid is sampled, and patient's supernatant chemokines CXCL9/MIG, CXCL10/IP-10 and CXCL11/ITAC are measured. A diagnosis of rheumatoid arthritis is diagnosed, if at least one of chemokines exceeds a threshold; the chemokine thresholds make 2625.8 pg/ml for CXCL9/MIG, 3108.2 pg/ml for CXCL9/MIG and 32.4 pg/ml for CXCL11/ITAC respectively. If the measured values are below the thresholds for three chemokines at the same time, the absence of rheumatoid arthritis and potential osteoarthrosis are stated.

EFFECT: using the given method enables differentiating rheumatoid arthritis and osteoarthrosis by measuring specific markers in the synovial fluid taken from the location directly.

2 ex

FIELD: medicine.

SUBSTANCE: invention relates to method of diagnosing rheumatoid arthritis, method of determining therapeutic agent for treatment of rheumatoid arthritis and set for realisation of methods. Methods are characterised by the fact that include stage of measuring amount of talin in plasma or serum of animal subject. Said measurement is carried out, for instance, by immunologic method with application of antibody, binding with talin. If amount of talin is higher than its average value in control subject without rheumatoid arthritis, rheumatoid arthritis is diagnosed in subject. In case of reduction of talin amount after introduction of therapeutic agent in comparison with amount of talin before introduction, therapeutic effect is stated. Set in accordance with claimed invention contains solid-phase carrier, to which antibody, binding with talin, is attached.

EFFECT: increased efficiency of diagnostics.

9 cl, 4 tbl, 4 ex, 3 dwg

FIELD: medicine.

SUBSTANCE: seromucoid concentration is measured in supernatant of a biological fluid aspirated from the nasopharynx of the newborns suffering a generalised form of the intrauterine mono-cytomegalovirus infection or mixed cytomegalovirus infection. If the seromucoid concentration is 0.110-0.140 absorbance units, the early stage of the generalised form of the intrauterine mono-cytomegalovirus infection is diagnosed. If the seromucoid concentration is 0.141-0.171 absorbance units, the early stage of the generalised form of the intrauterine mixed cytomegalovirus infection caused by a combination of the cytomegalovirus and type 1 herpes simplex virus is diagnosed.

EFFECT: using the declared method enables the effective differential diagnosis of the generalised form of the intrauterine mono or mixed cytomegalovirus infection in the newborns.

1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology. There are presented versions of a humanised anti-CD79b antibody, each of which is characterised by the presence of a light and heavy chain and a set of 6 CDR with a specified amino acid sequence and at least one free cysteine amino acid residue specified in A118C (according to the European Numeration) in the heavy chain and V205C (according to the Kabat numeration) in the light chain. There are disclosed: versions of a conjugate compound of the antibody and a drug preparation, wherein the antibody is bond to the drug preparation through free cysteine; an antibody-based pharmaceutical compound for treating cancer; method for detecting CD79b or cancer cells, as well as a method for inhibiting cell proliferation using the conjugate compound. What is described is a method for producing the conjugate compound.

EFFECT: invention can find further application in the therapy of CD79b-associated cancer diseases, including treating haemopoietic tumours in mammals.

70 cl, 20 tbl, 9 ex, 51 dwg

FIELD: medicine.

SUBSTANCE: fine-needle aspiration of nodular thyroid growths is controlled by ultrasonic examination. A puncture needle containing an aspirate is washed twice in normal saline 1 ml, centrifuged; a supernatant is selected, and thyroglobulin is measured by enzyme immunoassay. If the thyroglobulin content is less than 272.5 ng/ml, the absence of high differentiated cancer is stated; the value falling within the range of 272.5-355.5 ng/ml shows a risk of high differentiated thyroid cancer; if the thyroid value is more than 355.5 ng/ml, high differentiated thyroid cancer is suspected.

EFFECT: invention provides the pre-operative differential diagnostics of high differentiated cancer in the patients suffering nodular forms of thyroid diseases, and also enables the further selection of an adequate method of treating.

3 ex

FIELD: medicine.

SUBSTANCE: immunological indices in umbilical blood and venous blood on 4-5 day of life are determined. Resistance coefficient (Cres) is calculated by formula Cres=4.95258-0.0143955×(PI)+0.0962295×(PN)-0.00903362×(IgG)-0.257936×(IgA)-0.324514×(IgM)+0.430186×(CD45+CD3+)-1.66224×(CD45+CD4+CD3+)+l.49266×(CD45+CD8+CD3+)+0.815254×(CD4+CD8+)+0.522212×(HLA-DR+)-23.1991×(CD3+HLA-DR+)+0.974106×(CD3+CD25+)+0.832493×(CD3+CD4+CD25+)-3.52478×(CD16+CD56+CD3-)+7.67325×(CD16+CD56-CD3-)+11.082×(CD16+CD56+CD3+)+0.305366×(CD19+)+0.0691703×(CD5+)+0.0610707×(CD3+), where PI is phagocytic index, PN is phagocytic number, IgG, M, A are immunoglobulins of respective classes, CD are lymphocyte differentiation markers. If Cres value constitutes 1.78 and higher, when calculated by values of indices, obtained from umbilical blood, and is lower than 5.72, when counted by values of indices, obtained from venous blood on 4-5 day of life, on the first day of life high degree of resistance to infectious diseases is expected during the first year of life.

EFFECT: application of claimed method makes it possible to predict frequency of development of infectious diseases in child of the first year of life and take preventive measures to reduce risk of high morbidity.

2 tbl

FIELD: medicine.

SUBSTANCE: relative pre-implantation CD4+CD25+CD127- T-cell count is determined in the endometrial tissue of the females with primary and secondary infertility of an undefined origin. If the count is less than 7.5%, the immunological infertility is diagnosed. Using the given technique enables considering the CD4+CD25+CD127- T-cell deficiency as a marker of the immunological infertility in the females with both habitual miscarriage, and primary infertility.

EFFECT: improving the diagnostic procedure.

3 cl, 3 ex

FIELD: medicine.

SUBSTANCE: before prescribing contraception, polymorphous locus rs936306 of CYP19A1 gene is determined. If observing the genotype G/A detected, transient disorders for the first 6-6.5 months of the prescribed contraception is diagnosed; if observing the genotype A/A, side effects are diagnosed starting from the 7th months of the contraception.

EFFECT: technique according to the invention enables reducing a rate of side effects of the hormonal contraception, predicting the tolerance and safety of the hormonal contraceptives and the potential effect on the hepatic function, homeostasis system and blood lipids.

3 ex

FIELD: medicine.

SUBSTANCE: titre of IgM antibodies to cytomegalovirus (CMV) and titres of IgG antibodies to CMV in paired blood serums are determined in points (1 point - titres of IgM antibodies to CMV 1:200 and titres of antibodies IgG to CMV 1:200 - 1:400 with avidity index JgG to CMV higher than 65% in paired blood serums; 2 points - titre of IgM antibodies to CMV 1:400 and titres of IgG antibodies to CMV 1:400-1:800 with IgG avidity index to CMV higher than 65% in paired blood serums; 3 points - titre of IgM antibodies to CMV 1:400 and titres of IgG antibodies to CMV 1:200 - 1:800 with IgG avidity index to CMV higher than 65% in paired blood serums) (A), concentration of interferon -γ(pg/ml) in blood serum in the period of disease height of disease (B). After that, protracted course of acute rhinopharyngitis is determined by means of discriminant equation, including determined indices, D. If D value is lower than boundary value of discriminant function, absence of protracted course of acute rhinopharyngitis in women in case of reactivation of chronic cytomegaloviral infection in second trimester of pregnancy is predicted. If D is equal or is higher than boundary value, development of protracted course of acute rhinopharyngitis in women in case of reactivation of chronic cytomegaloviral infection in second trimester of pregnancy is predicted.

EFFECT: invention provides prediction of protracted course of acute rhinopharyngitis in case of reactivation of chronic cytomegaloviral infection in women in second trimester of pregnancy.

2 ex

FIELD: medicine.

SUBSTANCE: individual's peripheral blood is examined to measure the absolute content of natural killers (CD16+ lymphocytes) and the concentration of soluble apoptosis receptor (sAPO-1). If the CD16+ content is more than 0.5×109 cell/l, whereas the sAPO-1 concentration is more than 4,000 pg/ml, the risk of malignant new growths is predicted.

EFFECT: using the given method enables detecting the malignant new growth at the early stages with using immunological methods.

4 ex

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

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