Using glycosaminoglycans for decreasing non-specific binding in immunological assays

FIELD: medicine.

SUBSTANCE: invention represents an immunoassay reagent which contains an agent binding to an analyte in a diluent, and glycosaminoglycan in an amount sufficient to decrease non-specific binding in an analyte sample. In the presented immunoassay reagent, the analyte is troponin I binding to the analyte; the agent is a biotin-modified anti-troponin I antibody, and glycosaminoglycan is chondroitin sulphate. Also, the invention provides a composition containing the troponin I binding agent, and chondroitin sulphate in an amount sufficient to decrease non-specific binding in the troponin I sample. What is also provided is a method of detecting the analyte in the sample wherein non-specific binding is decreased by the use of glycosaminoglycan.

EFFECT: method improvement.

5 ex, 5 dwg

 

The technical FIELD

The present invention relates to immunological analyses, and, in particular, to the use of glycosaminoglycans in order to reduce nonspecific binding in immunological assays.

BACKGROUND of the INVENTION

Biochemical analyses linking widely used to determine the presence and concentration of the analyzed substances in biological samples. Such analyses are based on the concept of binding partners. Interested in an analyte associated with communicating with the analyzed substance by the agent (such as, for example, antibody to analyze the substance or receptor for the analyte). Thus, an analyte and binding to a target substance, the agent referred to as "binding partners". When one partner link associated with the solid phase, the analysis is called heterogeneous. Such heterogeneous assays include, for example, the sandwich method, the indirect method and the competitive method. All these terms are used in this technical field.

Sensitivity analysis usually means the smallest mass of analyte that provides a statistically significant change in the signal generated by the analysis, compared with the signal obtained in the absence of analyte. The increase in. the major is desirable because it can detect smaller amounts of analyte, and also provides higher overall accuracy of the measurement of the analyte.

Nonspecific binding means nonspecific interaction of the binding partners in a heterogeneous system analysis solid phase. The nonspecific binding is often reduces the sensitivity of heterogeneous assays, and it is therefore desirable to reduce this non-specific binding.

There are many methods used for this purpose. For example, proteins such as bovine serum albumin (BSA), gelatin and casein, is added to the reagents or pre-adsorb onto the solid phase, to block non-specific adsorption sites. In addition, the literature describes the use of various surfactants, often in high concentrations.

Although these methods can help a decrease in nonspecific adsorption, many of them are connected with the influence on the desired specific interaction partners bind. These methods can also lead to the replacement of the complex formed between the binding partners. In addition, despite the use of high concentrations of protein and surfactant, in many heterogeneous analyses there was a significant amount of nespa the specific binding. Thus, there is a need for alternative methods of reducing non-specific binding in heterogeneous assays.

This is especially true in the case of tests for determination of cardiac troponin I, where detectable levels of analyte are very small, and the increased sensitivity required to obtain accurate and useful results. Determination of cardiac troponin I helps in accurate diagnosis of acute myocardial infarction and risk stratification of patients with acute coronary syndromes without increase in ST segment in terms of the relative risk of mortality, myocardial infarction or increased probability of ischemic events requiring urgent revascularization procedures.

Troponin I (TnI) is a protein normally found in muscle tissue, and together with Troponin T and Troponin With regulates calcium-dependent interaction of actin and myosin (Tobacman, Annu Rev Physiol 58:447-481, 1996). Identified three isotype TnI: one associated with a fast twitch skeletal muscle, and the other with slow twitch skeletal muscle, and a third cardiac muscle (Wilkinson and Grand, Nature 271:31-35, 1978; Bodor, J Clin Immunoassay 17(l):40-44, 1994). The heart shape has 31 additional amino acid residue at the N-end and is the only isoform of troponin, prisutstvie the processes in the myocardium (Vallins et al., FEBS Letts 270(1,2):57-61, 1990).

Clinical studies have demonstrated that cardiac troponin I (cTnI) is detected in the bloodstream after 4-6 hours after acute myocardial infarction (AMI) and remains elevated for several days after (Mair et al., Clin Chem 41(9):1266-1272, 1995; Lame et al., Clin Chem 39(6):972-979, 1993). Thus, raising the level of cTnI covers diagnostic window of creatine kinase-MB (ck-MB) and lactate dehydrogenase (Bodor, J Clin Immunoassay 17(l):40-44, 1994). Further research indicated that cTnI has a higher clinical specificity for myocardial injury than ck-MB (Adams et al., Circulation 88(1): 101-106, 1993; Apple et al., Clin Chim Acta 237:59-66, 1995).

Due to its cardiac specificity and sensitivity TnI is used as a reliable marker in the evaluation of patients with unstable angina and acute coronary syndrome without increasing the ST segment (ACS). Previous clinical studies of patients with ACS (Lindahl et al., J Am Coll Cardiol 38:1497-1498, 2001; Venge et al., Am J Cardiol 89:1035-1041, 2002) showed that a slight increase of cTnI values provides important prognostic information about the near and long-term risk of death (Galvani et al., Circulation 95:2053-2059, 1997; Antman et al., N Eng J Med 335:1342-1349, 1996; Ottani et al., Am Heart J 40:917-927, 2000; Heidenreich et al., J Am Coll Cardiol 38:478-485, 2001). Ultimately, the evaluation of the forecast can be useful to identify patients for whom, most likely, will be useful for certain those who piticescu intervention.

Thus, it is desirable to obtain reagents and methods to reduce nonspecific binding in heterogeneous assays for cTnI, a means of increasing the sensitivity analyses for cTnI.

SUMMARY of the INVENTION

For this purpose the invention provides immunological analysis reagent comprising communicating with the analyzed substance agent in the diluent and glycosaminoglycan in a quantity sufficient to reduce nonspecific binding in the analysis of the sample analyte.

In one preferred in the present embodiment, the analyzed substance is troponin, communicating with the analyzed substance agent - antitripsin I monoclonal antibody, and glycosaminoglycans - chondroitin sulfate.

Also provided the composition of the sample, which includes a sample for analysis for the presence of the analyte binding to the target substance to the agent and glycosaminoglycan other than heparin, in a quantity sufficient to reduce nonspecific binding in the analysis sample for an analyte.

In one preferred in the present embodiment, the breakdown is serum or EDTA plasma, analyzed substance - troponin binding to the target substance agent - antitripsin I m nacionalne antibody and glycosaminoglycans - chondroitin sulfate.

Also provides methods of detecting an analyte in a sample using fractions to reduce nonspecific binding in the way. The method comprises combining the sample analyzed for the presence of the analyte, with glycosaminoglycans and communicating with the analyzed substance agent for the formation of a complex between the analyzed substance in the sample and communicating with the analyzed substance agent, and glycosaminoglycan reduces the nonspecific binding in the method and detecting the resulting complex for determination of the analyte. In this preferred method the analyzed substance is troponin, more preferably troponin I, a preferred glycosaminoglycans is chondroitin sulfate.

Additional features and advantages of this invention will become clear from the following description which should be read in conjunction with the attached drawings.

BRIEF DESCRIPTION of DRAWINGS

In Fig. 1 shows the results of the analysis TropI when various sugars were introduced into the sample TropI invalid results;

In Fig. 2 shows the results of the analysis TropI when the reagent BJ was prepared from chondroitin sulfate at 0, 1, 2 and 4 mg/ml in the presence or without the AI EDTA;

In Fig. 3 shows the results of the analysis TropI with the addition of 0.5 mg/ml of CSC in the reagent BJ and without such additions;

In Fig. 4 shows the results of the analysis TropI when the reagent BJ was prepared with different isomers of chondroitin sulfate compared with Kit Lot (without chondroitin sulphate); and

In Fig. 5 shows the principles of analysis of cardiac troponin I.

DETAILED description of the INVENTION

The invention provides immunological analysis reagent, which includes communicating with the analyzed substance agent in the diluent and glycosaminoglycan in a quantity sufficient to reduce nonspecific binding in the analysis sample for an analyte.

As discussed above, it is often desirable to determine the presence and concentration of the analyzed substances in biological samples. An analyte is a substance or chemical component, which is determined during the analytical procedure (such as immunological analysis). Immunological analyses are based on the concept of binding partners. Interest an analyte associated with communicating with the analyzed substance by the agent (such as, for example, antibody to analyze the substance or receptor for the analyte). Thus, an analyte and binding with an analyte is the agent referred to as "binding partners".

In many immunological assays to bind to the target substance, the agent is an antibody. Such antibodies are often provided in the diluent, such as califofnia buffer. The antibody can be from any class of immunoglobulins, including IgG or IgM. The antibody may be a monoclonal antibody or a polyclonal antibody. In sandic-immunoassay an analyte can be captured by the antibody or antibody immobilized on the solid phase. Such immobilization can be achieved using methods known from the prior art, including the use of streptavidin coated (SAC) of the solid phase, is connected with the Biotin-labeled antibody or antibodies. Interest an analyte present in the sample binds to the immobilized antibody capture or antibodies, and then labeled antibody or antibodies, in turn, are associated with the captured target substance. The label can be any known in the prior art, including, for example, horseradish peroxidase and alkaline phosphatase. The detected signal indicative of the amount of analyte present in the sample. The method of detection will depend on the type of the selected tag, as known from the prior art, and may include colorimetric, fluorometric is or chemiluminescent methods.

The currently favored by glycosaminoglycans (GAG is chondroitin sulfate, although they may also use other GAG. Other GAG include hyaluronate (also called hyaluronic acid), heparansulfate, heparin, dermatologit and keratinolytic. The chondroitin sulfate may be chondroitin sulfate A, chondroitin sulfate B (also called dermatologit), chondroitin sulfate or a mixture thereof.

Glycosaminoglycans (GAG) or mucopolysaccharides are long unbranched polysaccharides containing repeated disaccharide glycosides motive. Disaccharide glycosides motifs contain one or two modified sugars, N-atsetilgalaktozamin (GalNAc) or N-acetylglucosamine (GlcNAc) and a uronic acid such as glucuronate or iduronate. The hyaluronates consist of D-glucuronate and GlcNAc. Dermatosurgery composed of D-glucuronic acid (GlcA) or L-iduronate (IdoA) and GalNAc-sulfate. Heterogeneity in dermatosurgery is the result of different degrees of O-sulfate crystallization and the presence of two uronic acids. The chondroitin sulfates consist of D-glucuronate and GalNAc-6 (or 4)-sulfate. Heparin and heparansulfate consist of D-glucuronate-2-sulfate and N-sulfo-In-glucosamine-6-sulfate (heparan contain less sulfate than heparins). Createsurface composed of galactose and galactose-6-sulfate and GlcNAc-6-sulfate.

Chondroitin sulfate CS) is sulfated by glycosaminoglycans (GAG). In the chain of chondroitin may be more than 100 individual sugars, each of which can be sulfated in different positions and quantities. Chondroitin sulfate And means CS predominantly sulfated byto 4 carbon sugar GalNAc (chondroitin-4-sulfate). Chondroitin sulfate In call dermatosurgical. Chondroitin sulfate With a mean CS, predominantly sulfated at carbon 6 of the GalNAc sugar (chondroitin-6-sulfate). Chondroitin sulfate D means CS, predominantly sulfated at carbon 2 of GlcA and 6 of the GalNAc sugar (chondroitin-4,6-sulfate).

Glycosaminoglycan is provided in a quantity sufficient to reduce nonspecific binding in the analysis sample for an analyte. If GAG is chondroitin sulfate, this amount preferably ranges from about 0.25 mg/ml to about 4 mg/ml (equivalent to approximately of 0.025% to about 0.4%). In one specific embodiment, the chondroitin sulfate is present in quantities of 1 mg/ml (equivalent to 0.1%). The following examples illustrate in detail the selection of the appropriate number of GAG for use in accordance with this invention.

Analyzed for the presence of the analyte sample may be any suitable sample, preferably a breakdown of the blood, such as the sample is serum or plasma. Plasma cu is VI - the liquid component of blood, in which the suspended blood cells. Centrifugation is a simple way of separating plasma from blood cells in the blood sample. Serum is the blood plasma, from which the factors of the blood coagulation system have been removed in a natural way, allowing the blood to clot to release the liquid component. Plasma samples obtained from test tubes with blood containing anticoagulants such as heparin sodium, sodium citrate, sodium fluoride and potassium oxalate or potassium EDTA (ethylenediaminetetraacetic acid). In the case of plasma samples according to this invention, the plasma is preferably receive, using an anticoagulant other than heparin.

In one embodiment, the immunological analysis reagent according to this invention includes a monoclonal antibody specific to cardiac troponin I in the diluent and 0.1% chondroitin sulphate. Suitable antibodies to cardiac troponin I is known from the prior art, and a specific pair or combination of antibodies is often recommended as partners for analysis. In particular, can be used monoclonal antibodies, denoted S and 24-40 as double antibody capture, labeled with Biotin, for connection to a streptavidin coated well, and antibody A, as antibody detection of peroxidase labeled are you the and. These antibodies are commercially available (see sources listed below) and discussed in the literature in connection with tests for cardiac troponin I, and procedures biotinidase and tagging is also known from the prior art.

The above description relates to the immunological analysis reagent, which includes glycosaminoglycan in a quantity sufficient to reduce nonspecific binding. GAG may be present in the diluent containing the antibody. Alternatively, the GAG can be added to the composition of the sample, which is then added to bind to the target substance to the agent. The procedure of preparation may vary, but the GAG should be added before the onset of nonspecific binding of any analyte present in the sample, to bind to the target substance by the agent.

Thus, a composition of the sample, which includes a sample for analysis for the presence of the analyte binding to the target substance to the agent and glycosaminoglycan other than heparin, in a quantity sufficient to reduce nonspecific binding in the analysis sample for an analyte.

In one preferred in the present embodiment, the breakdown is serum or EDTA plasma, analyzed substance - troponin, communicating with analiziruemykh substance agent antitripsin I monoclonal antibody, and glycosaminoglycans - chondroitin sulfate.

Also proposes a method of detecting an analyte in a sample, comprising: combining analyzed for the presence of the analyte sample with glycosaminoglycans and communicating with the analyzed substance agent for the formation of a complex between the analyzed substance present in the sample, and communicating with the analyzed substance agent, and glycosaminoglycan reduces the nonspecific binding in the way; and detecting the resulting complex for determination of the analyte. In one embodiment, the sample is combined with glycosaminoglycans and the resulting sample is then combined with communicating with the analyzed substance by the agent. In another embodiment, the sample is combined with communicating with the analyzed substance agent, and the resulting sample is then combined with glycosaminoglycans. In yet another embodiment, communicating with the analyzed substance agent obtained as reagent immunoassay, including communicating with the analyzed substance agent in the diluent and glycosaminoglycan, and immunological analysis reagent combines with the sample. Each of these methods, preferred an analyte - troponin, more FAV is preferably - troponin I, and the preferred glycosaminoglycans is chondroitin sulfate.

In the composition of the sample and methods according to this invention, various suitable communicating with the analyzed substance agents, thinner, glycosaminoglycans, sample and analyte are as described above in connection with reagents for immunological analyses.

The reagents, compositions and methods of the present invention is particularly useful in the immunological analysis of cardiac troponin I. the Details of this version of the implementation is given in the following examples.

Example I. the Impact of adding heparin to the capture reagents (BJ) and detection (CJ) cardiac troponin I (cTnI or TropI) analysis

The purpose of this experiment was to determine whether the addition of heparin to the reagents TropI BJ and/or CJ reduction of false positive results TropI. Received numerous reports of reproducible false results of increased Troponin I in serum samples. Also there have been several reports falsely elevated TropI plasma EDTA. In some cases, the corresponding plasma samples with heparin, taken from the same patients did not give false results elevated TropI.

The experiment included the introduction of heparin in the reagents BJ and CJ and analysis with the use of these reagents ol the b TropI, which previously gave false positive results TropI.

The results show that the introduction of heparin in TropI CJ and immediate analysis of the observed strong attenuation of the received signal. With only 10 units per ml CJ received signal Cal 2 (calibration 2) was less than 50% of nominal. Levels of heparin in plasma, in contrast, are usually 25-50 units/ml To ensure equivalent concentration of heparin, provided by the reagent CJ would require the presence of heparin at the level of 50-100 units/ml On the basis of these responses is not possible to add heparin to CJ at levels that would be equivalent of heparinized plasma.

A group of serum samples, which previously gave false positive results on TropI, was tested using the solution CJ containing 10 units of heparin on These ml. gave a false test results demonstrated a significant reduction in detectable concentrations of cTnI (cardiac troponin I). The results were in the range of 8-35% for samples, which without treatment showed troponin I concentrations of 0.7-7.0 ng/ml, However, none of these samples failed to fully adjust to a level below the Upper reference limit (URL) for serum.

The results of this experiment lead to the conclusion that the addition of heparin to the composition CJ PR which leads to a significant reduction in the overall ability of the reagents to generate the signal. At concentrations of heparin 10 units per ml, the signal was only 50% of nominal. However, giving false results of the patient samples were partially corrected by adding the specified amount of heparin to CJ. However, a more complete correction of the specified effect would require higher levels of heparin, which likely would reduce the signal analysis to levels incompatible with the principles of analysis.

Example II. Influence of sugars as the adjustment factors of the sample in the reagent BJ

The objective of this experiment was to evaluate the ability of sugars, such as sugars present in glycosaminoglycan and is usually associated with the side chains of carbohydrate horseradish peroxidase (HRP), to reduce the impact of false-positive samples TropI adding reagents BJ.

As described in Example I, the addition of heparin to false-positive samples decreased false positive results. Heparin is a heterogeneous group of anionic mucopolysaccharides with unbranched chain called glycosaminoglycans (GAG), which have anticoagulant properties. Although there might be other sugars, the main sugars in heparin are: α-L-iduronovoy acid 2-sulfate; 2-deoxy-2-sulfamido-α-D-glucose 6-sulfate; β-D-glucuronic acid; 2-acetamido-2-deoxy-α-D-glucose; and α-L-is doranova acid.

As an initial study of possible adjustment factors sample in false-positive tests TropI have introduced the following sugar (to achieve final concentration of 2 mg/ml of test substance to evaluate the effectiveness of blocking false positive reactions: N-acetyl-glucosamine (Sigma A8625); N-acetyl-galactosamine (Sigma A2795); glucosamine (Sigma G4875); N-acetylneuraminic acid (NANA, sialic acid)(Sigma A0812); chondroitin sulfate C (Sigma C4384); chitin (a homopolymer of N-acetyl-glucosamine) (Sigma C9752); the mucin (polymer NANA)(Sigma M3895); and mannose (Sigma M8296). In this initial study, it was found that chondroitin sulfate C (CSC) significantly reduces false positive results (see Fig. 1). The results TropI were generally suppressed to below URL in false-positive samples, except one. We believe that this last test positive for heterophile antibodies.

Based on these initial qualifying studies were conducted additional experiments in which chondroitin sulfate C (CSC) was added directly to the reagent BJ in a series of increasing levels(0,25, 0,5, 1, 2, 3 and 4 mg/ml), with and without EDTA (to 5.58 mg/ml) reagent BJ (see Fig. 2 and 3). The effectiveness of the compositions was based on the blocking of false-positive samples TropI in combination with the assessment of changes in response reference calibrators low concentration of CSC, which effectively suppressed false positive samples TropI was 0.25 mg/ml; at slightly higher levels CSC (0.5 mg/ml) was observed for some of the increasing improvement of blocking. The effect associated with the presence or absence of EDTA with added reagents, was not significant.

Add CSC in the amount of 0.25-0.5 mg/ml of the composition BJ had a small but noticeable effect on the negative or positive control serum samples. In addition, the feedback reference calibrator showed little change at low levels CSC. At levels of CSC in excess of 1 mg/ml is usually observed 10-30% reduction in response.

The results of this experiment lead to the conclusion that chondroitin sulfate is an effective blocking agent to reduce non-specific background reaction (nonspecific binding), which show some samples of serum and EDTA plasma. While the lowest tested level (0.25 mg/ml) showed a significant reduction in the observed false-positive reactions, slightly higher levels (0.5 mg/ml) may provide additional protective measures for samples that may contain higher levels of the interfering substance. The level of EDTA in the presence of CSC had no significant effect on the false positive samples.

Example III. False-positive samples with 0.05% of CSC in BJ

The objective of this experiment was to test false positive samples using 0.5 mg/ml (0.05%) of CSC in BJ. False positive samples gave positive results without adding CSC and negative adding 0.05% of CSC to bitenova the diluent. The addition of 0.05% CSC had no effect on the prediction of a positive or negative result of the test. These results are shown in Fig. 3.

Example IV. Comparative indicators of chondroitin a, b and C

The purpose of this experiment was to determine whether any of the isomers of chondroitin sulfate to provide protection from receiving false test results. Chondroitin sulfate, which was used in the above experiments was a mixture of chondroitin sulphate and With a certain amount of chondroitin sulfate A. the Least amount of chondroitin sulfate in a mixture of 85%. Because there can be up to 15% of the isomer And efficiency were tested isomers a, b and C.

The results showed that all isomers of chondroitin sulfate was successfully reduced the observed concentrations in false-positive samples, and there were no significant differences between the isomers in relation to the suppression of false-positive reactions (see Fig. 4). This leads to the conclusion that the percentage purity of chondroitin sulfate in connection with the concentrations of the isomers a and b does not affect the suppression of false-positive responses.

Example V a Sample of cardiac troponin I

Used the method immunometric immunological analysis (see Fig. 5), including simultaneous reaction of cardiac troponin I present in the sample, with biotinylating antibodies (mouse monoclonal anti-cTnI: clone S that recognizes amino acids 41-49 Troponin I, and the second clone, specific for the site of Troponin I comprising amino acids 24-40) and conjugate antibodies labeled with horseradish peroxidase (HRP) (mouse monoclonal anti-cTnI: clone A that recognizes amino acids 87-91 Troponin I). Biotinylated than that of tri test Mab react with specific troponin I in a sample with the formation of the complex to bind with streptavidin wells SAC. Unbound materials are removed by washing, and the troponin complex find using HRP-labeled Mab (specific binds to the epitope of troponin I, which differs from the epitope with which the tie is by biotinylated Mab). Bound HRP conjugate is measured by a luminescent reaction. The reagent containing luminogenic substrates (derived lyuminola and salt percolate) and the agent of the transfer of electrons added to the wells. HRP related conjugates catalyzes the oxidation of the derivative lyuminola, producing light. Agent transfer electrons (substituted acetanilide) increases the level of light produced and prolongs its radiation. The light signals are read immunodiagnostics system VITROS™ (Ortho-Clinical Diagnostics, Inc., Raritan, New Jersey). The amount of bound HRP conjugate is directly proportional to the concentration of cTnI present.

Protocol analysis was as follows: 1) the hole in the SAC was added 80 μl of the sample, 35 μl bioteknologi reagent (BJ) and 35 ál of conjugate (CJ); 2) Incubation for 10 minutes and 40 seconds; 3) Washing the cell SAC; 4) Add 200 ál of the signal and measurement of the emitted light.

Wells SAC was prepared by coating the polystyrene wells with streptavidin. Briefly, polystyrene microtiter wells were first irradiated to 3.5 Mrad to optimize the adsorption of proteins. Biotinylated bovine serum albumin (B-BSA), obtained by chemical compounds Biotin to BSA using commercially available activated ester (Biotin-XX-NHS, Calbiochem, Nottingham, UK), were applied to the polystyrene wells. The coating was applied by inquire the project for wells with a solution of B-BSA for 10 minutes. B-BSA was physically adsorbiroval and not covalently associated with the surface of the polystyrene. The wells were washed before coating of streptavidin. Streptavidin was applied to the wells by incubation streptavidin solution with Biotin surface for 50 minutes. The interaction between streptavidin and Biotin is non-covalent, but very durable (1015l/mol). The wells were washed again and then dried and stored.

Streptavidin has four binding site with Biotin, so after immobilization of streptavidin on the surface of vacant plots of binding with Biotin. These binding sites can react with biotinylating components analysis.

The conjugate reagent (CJ) includes the following components:

Component Number g/l

ComponentThe number of g/l
Water849,3
To2NRA413
KN2RHO417
Kathon20
BSA 30%100
HRP-labeled mab 16A11 CL is n 4 mg/l
pH6,6

Reagent analysis or capture (BJ) includes the following components:

ComponentThe number of g/l
Water
To2NRA413
KN2RHO417
Kathon20
Bovine serum albumin 30%100
EDTA (equimolecular disodium and chinatravel)15 mm
Chondroitin sulfate1
Biotinylated mab 24-40aa-specific clone5,5 mg/l
Biotinylated mab clone 19C73 mg/l
pH6,6

Monochloramine antibodies are available commercially. HyTest Ltd (Itainen Pitkakatu 4C, Pharma City, Turku, 20520 Finland) is a provider of clone S murine monoclonal anti the La, specific to the field of troponin I comprising amino acids 41-49. This Mab biotinylation, as described below. HyTest also supplies a clone A mouse monoclonal antibodies specific to the field of troponin I comprising amino acids 87-91. This Mab mark HRP, as described below. Strategic BioSolutions (111 Pencader Dr., Newark, Delaware, 19702 USA) delivers a clone of murine monoclonal antibodies specific to the field of troponin I comprising amino acids 24-40. This b biotinylation, as described below.

Procedure biotinidase includes the following. Clone S and directed clone Strategic BioSolutions 24-40 kongugiruut with Biotin separately through a well-known region-specific chemistry.

The procedure of HRP labeling includes the following. Clone A from HyTest kongugiruut with HRP using the following methodology: 1) Mab activate maleimide groups by the reaction with sulfo-SMCC [sulfosuccinimidyl 4-(M-maleimidomethyl) cyclohexane-1-carboxylate]; 2) HRP activate thiol groups by the reaction with NHS-SATA [N-hydroxysuccinimide s-acetylthiocholine acid]; C) Both activated reagent is purified and then spend their reaction with the formation of A-HRP, which is then purified.

Although there is described the specific embodiments of the invention, it should be understood that the invention is not limited to, a specialist in this on the region of the equipment may make various changes, in particular, in light of the above description. In the following description it is possible to make reasonable changes and modifications without leaving the scope of the invention.

1. The immunological analysis reagent, including
bind with troponin I agent in the diluent and chondroitin sulfate in a quantity sufficient to reduce nonspecific binding in the analysis of samples for troponin I.

2. The immunological analysis reagent according to claim 1, wherein the chondroitin sulfate is present in an amount of from about 0.25 mg/ml to about 4 mg/ml

3. The immunological analysis reagent according to claim 1, wherein the chondroitin sulfate is present in amount of about 1 mg/ml

4. The immunological analysis reagent according to claim 1, wherein the communicating with troponin I, the agent is an antibody.

5. The composition of the samples for immunological analysis, including
the sample analyzed for the presence of troponin I; bind with troponin I agent; chondroitin sulfate in a quantity sufficient to reduce nonspecific binding in the analysis of samples for troponin I.

6. The composition of the sample according to claim 5, characterized in that the sample is a serum sample.

7. The composition of the sample according to claim 5, characterized in that the sample is a plasma sample containing ethylenediaminetetraacetic sour is from.

8. The composition of the sample according to claim 5, characterized in that bind with troponin I, the agent is an antibody.

9. The method of detection of troponin I in a sample comprising combining analyzed for the presence of troponin I samples with chondroitin sulfate and bind with troponin I agent for the formation of a complex between troponin I present in the sample, to bind with troponin I by the agent, and chondroitin sulfate reduces the nonspecific binding in the way; and detection of the resulting complex to determine troponin I.

10. The method of claim 9, wherein the sample is combined with the chondroitin sulfate, and the resulting sample is then combined with communicating with troponin I by the agent.

11. The method according to claim 9, wherein the sample is combined with communicating with troponin I by the agent, and the resulting sample is then combined with the chondroitin sulfate.

12. The method according to claim 9, wherein the communicating with troponin I, the agent obtained as reagent immunoassay, including communicating with troponin I agent in the diluent and chondroitin sulfate, and the fact that the immunological analysis reagent together with a breakdown.



 

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6 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.

EFFECT: method provides quick identification and high-reliability differentiation of all strains.

7 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: set of reagents for quantitative determination of avermectins contains an ivermectin conjugate with bovine serum albumin which is immobilised on a polystyrene dish, a peroxidase conjugate of highly specific mouse monoclonal antibodies to ivermectin, and ivermectin calibration samples (with concentration between 0 and 100 ng/ml). The monoclonal antibodies in the set are produced by a strain of hybrid cultured cells of Mus. Museums L., which are deposited in the Collection of transferred vertebrate somatic cells of the D. I. Ivanovsky Research Institute of Virology under No. 05/09. From non-specific components, the set contains a buffer for culturing the monoclonal antibody conjugate with horseradish peroxidase, a buffer for culturing the analysed samples and washing the trays, reagents for detecting peroxidase activity, a substrate solution, hydrogen peroxide and tetramethylbenzidine and a stop solution (1M sulphuric acid). The disclosed set is designed to detect residual quantities of avermectins in tissue and biological fluids for monitoring chemical contamination of animal products.

EFFECT: use of the set enables to determine main components of an avermectin complex in animal products, while providing high sensitivity and specificity of detection.

1 dwg, 2 tbl, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention discloses a monomer single-strand V93 nanoantibody which can bind and inhibit the human vascular endothelial growth factor. The invention describes a nucleotide sequence which codes the V93 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V93 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V93 nanoantibody, as well as use of the V93 nanoantibody for qualitative and quantitative determination of VEGF in a sample.

EFFECT: use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

7 cl, 7 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention describes a nucleotide sequence which codes the V9 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V9 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V9 nanoantibody, as well as use of the V9 nanoantibody for qualitative and quantitative determination of VEGF in a sample. Use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

EFFECT: invention discloses a monomer single-strand V9 nanoantibody which can bind and inhibit the human vascular endothelial growth factor.

7 cl, 7 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: immunological detection and quantitative analysis of sequential changes in protein levels of VEGF-165 in obtained patient's samples taken in course of time are conducted, where increasing levels of the protein VEGF-165 in course of time indicate progression of disease or adverse reaction to the therapy, and where decreasing levels of protein VEGF-165 in course of time indicate remission of the disease or a positive reaction to the therapy.

EFFECT: method enables to conduct noninvasive analysis of levels of circulating VEGF-165 which serves as a valuable prognostic indicator of disease outcome.

22 cl, 2 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method involves evaluating proliferating processes by calculating index of positive cell nuclei (Ki-67). The Ki-67 value being from 6 to 16%, erosive ulcerating stomach lesions accompanied by stomach hemorrhage and hemorrhagic shock is to be predicted. The value being from 17 to 30%, erosive ulcerating stomach lesions without hemorrhage is to be predicted.

EFFECT: high accuracy of prognosis.

1 tbl

FIELD: medicine, virology.

SUBSTANCE: invention relates to diagnosis of infection caused by the Epstein-Barr virus. Method involves carrying out the indirect immune peroxidase reaction followed by histochemical staining and detection of the Epstein-Barr virus antigen. Additionally, method provides diagnosis of severity for the infectious mononucleosis course based on the expression of Epstein-Barr virus antigen. Invention provides enhancing precision in diagnosis of infection caused by the Epstein-Barr virus and assay for severity in course of the infectious mononucleosis.

EFFECT: improved diagnosis method.

1 tbl, 2 ex

FIELD: medicinal biochemistry.

SUBSTANCE: the present innovation deals with detecting oncoprotein E7 of human papilloma virus (HPV) in biopsy sample with the help of the pairs of monoclonal antibodies referring to IgG2a and IgG2b groups chosen out of the following groups: 716-321, 716-325, 716-332, 716-343, 716-281, 716-288 one of which is indicated for primary protein binding and another, being the antibody conjugate with enzymatic label - to detect the complexes developed.

EFFECT: higher sensitivity of the method.

5 cl, 4 dwg, 4 ex, 2 tbl

FIELD: medicine, therapy, obstetrics.

SUBSTANCE: at gestation terms of 20-28 wk in peripheral blood one should detect the index for the ratio of relative content of CD4+ to CD8+ lymphocytes. At values of CD4+/CD8+ being equal to or below 2.4 it is possible to predict positive effect of common therapy, and at values being above 2.4 one should predict thorough and prolonged therapy. The method enables to match another therapeutic tactics in due time.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine, obstetrics.

SUBSTANCE: one should detect during pregnancy-free period relative content of CD38+ lymphocytes in peripheral blood, at its value being either equal to or above 12% one should predict efficient restoration of reproductive function. The method is very simple in application.

EFFECT: higher accuracy and sensitivity of detection.

3 ex, 1 tbl

FIELD: medicine, pediatrics.

SUBSTANCE: in 5-10-d-aged neonatals one should detect relative content of CD8+HLA-DR+ lymphocytes in peripheral blood and at its value being below 2.4% it is possible to predict the healing at different types of neonatal infections.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine, gynecology.

SUBSTANCE: invention relates to a method for diagnosis of internal endometriosis in peripheral venous blood of women wherein the relative content of lymphocytes CD25+ is determined. Internal endometriosis is diagnosed at values of this index 6% or above. Proposed method provides carrying out diagnosis of internal endometriosis in women with high precision, sensitivity and specificity that allows carrying out the correct and well-timed necessary complex of curative-prophylactic treatment.

EFFECT: improved method for diagnosis.

1 tbl, 3 ex

FIELD: medicine, immunological laboratory diagnostics.

SUBSTANCE: at terms from 6 to 12 wk of gestation one should study relative content of CD3+CD16+ lymphocytes in peripheral venous blood and at its values being either equal or above 5.4% one should predict the development of light-degree gestosis to carry out the complex of curative-prophylactic means.

EFFECT: higher efficiency of prediction.

3 ex, 1 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: during the 1st trimester of pregnancy (6-13 wk) in peripheral venous blood in women at risk of failed pregnancy one should detect relative content of CD16+CD56- lymphocytes and at its value being either equal or below 11% it is possible to predict the development of infectious diseases in full-term neonatals during the first 7-10 d of their lives. The innovation enables to predict the development of local form of infectious-inflammatory diseases in full-term neonatals.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine, clinical laboratory diagnostics.

SUBSTANCE: in the sample of peripheral venous blood one should determine relative content of CD45RO+ lymphocytes and at its value being equal to 31% or lower it is possible to diagnose external genital endometriosis. The method is atraumatic and enables to diagnose external genital endometriosis at high accuracy.

EFFECT: higher efficiency of diagnostics.

3 ex, 1 tbl

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