Methods, devices and sets for detection or monitoring of acute kidney injury

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and deals with a method of determining in an individual's sample of a protein of neutrophilic origin, gelatinase-associated lipocalin (NGAL), for determination of acute kidney injury in the individual, where the predominating amount of monomer and/or heterodimer forms of NGAL protein, in comparison with the dimer form of NGAL protein, shows that NGAL protein originates from the individual's kidneys and that the individual has acute kidney injury, whereas equal or predominating amount of the dimer form of NGAL protein, in comparison with the monomer or heterodimer form of NGAL protein shows that NGAL protein originates from the individual's neutrophils and that the said individual does not have acute kidney injury; a set for determination of relative amounts of monomer, dimer or heterodimer forms of NGAL; application of the device in the said method.

EFFECT: group of inventions ensures more accurate diagnosis and therefore contributes to better directed treatment.

17 cl, 2 ex, 10 dwg, 1 tbl

 

The scope of the invention

The present invention relates to methods, devices and kits for the detection or monitoring of acute kidney injury and specifically to such methods and kits that are based on the measurement of lipocalin, a protein associated with gelatinase neutrophils (NGAL), also known as lipocalin human neutrophils (HNL).

Background of the invention

Acute kidney injury (AKI) is a serious condition that can develop as a postoperative complication, for example, as a complication after heart surgery or kidney transplantation, as a side effect after the introduction of diagnostic toolsin vivofor example, contrast media or nephrotoxic therapeutic agents, etc. and/or as a result of other medical conditions such as diabetes, septicemia, hemorrhagic shock, and the like. For example, acute kidney injury may occur in 30% of all patients after surgery on the heart, and is associated with high mortality, a more complicated course of treatment, patients receiving dialysis with a reduced quality of life and a high risk of infectious complications. Although it is known that treatment in the early stages of developing acute renal failure reduces mortality and/or reduce the em period of treatment, in the early stages it is often difficult to determine the presence of acute renal failure.

In clinical practice, currently used, the standard diagnosis of AKI was named RIFLE (emergence, injury, failure, loss of function, end-stage disease), which is based or elevated creatinine in blood serum, or decreased urine output. Creatinine in serum is a reliable marker of the state of the overall function of the kidneys, but in severe renal impairment creatinine levels is unreliable and changing its level is delayed.

Fortunately, discovered several promising biomarkers, including HNL/NGAL (in the description designated as NGAL), a molecule kidney damage-1, cystatin C and IL-18.

Special attention is focused on the use of NGAL protein as a marker of acute kidney injury. NGAL is a glycoprotein and was originally discovered as a specific component of the granules of neutrophils and is a member of lipocalin family of proteins. It is shown that the protein exists as a monomer with a mass of 25 kDa, and as linked by a disulfide bond glycosilated with a mass of 45 kDa, and can also be covalently linked to gelatinases neutrophils (also known as matrix metalloproteinase 9, MMP-9) via intermolecular disulfide bridge and with whom to exist in the form heterodimeric form with a mass of 135 kDa. NGAL first described as HNL, as a specific marker of activity of neutrophilsin vivoandin vitroXuet al,Journal of Immunological Methods, 171:245-252 (1994) and for use as a diagnostic marker of inflammation, Venge, U.S. patent 6136526, which is incorporated herein by reference.

Later Devarajanet althe patent publication U.S. No. 2004/0219603 A1 and 2005/0272101 A1 describe the use of NGAL as a biomarker of damage to renal tubular cells and other diseases and kidney damage. Recently to diagnose early renal failure BioBorto Diagnostics, Gentofte, Denmark suggested “NGAL ELISA Kit, as well as mouse monoclonal antibodies against human NGAL and mouse monoclonal antibodies against rat NGAL. Advanced Dentet al, Critical Care, 11(6):R127 (2007) described the device Triage® NGAL from Biosite Inc., San Diego, CA, using specific against NGAL monoclonal antibody conjugated with a fluorescent nanoparticle for use in the measurement of NGAL as a biomarker of acute kidney injury.

However, it is necessary to further improve the detection and/or monitoring of acute renal failure.

The invention

Thus, the present invention relates to methods, devices and kits for detection of acute kidney injury and for monitoring the effectiveness of treatments for acute damage the statement of the kidneys.

In one of the embodiments the invention relates to a method for determining acute kidney injury in an individual, where the method comprises (a) bringing into contact a sample of body fluid of an individual with a device for analysis, where the device includes an antibody to the associated with gelatinase of neutrophils to lipocalin (NGAL) and detektiruya tag for the possibility of complex formation between protein NGAL in the sample and the antibody to NGAL and (b) determining the amount of complex formed between NGAL protein and an antibody against NGAL in the device for analysis, using a detectable label, where the antibody to NGAL in the device has the ability to bind more than two epitopes of the protein NGAL and where the amount of formed complex reflects the severity of acute kidney injury.

In another embodiment, the invention relates to a method for the detection of acute kidney injury in an individual, where the method comprises (a) bringing into contact of the sample of body fluid with a polyclonal antibody to the associated with gelatinase of neutrophils to lipocalin (NGAL) and (b) determining the amount of complex formed between NGAL sample and a polyclonal antibody to NGAL, using a detectable label, where the number of complex reflects the severity of acute kidney injury.

In another embodiment, the invention relates the method of monitoring the efficacy of treatment of acute kidney injury where the method includes the stage of (a) bringing into contact of the first sample of body fluid with the first device for analysis, comprising the antibody to the associated with gelatinase of neutrophils to lipocalin (NGAL) and detektiruya tag for the formation of a complex between the protein NGAL in the first sample with the antibody to NGAL, (b) determining the amount of complex formed by protein NGAL in the first sample and the antibody to NGAL in the first device for analysis, using a detectable label, where the antibody to NGAL in the device has the ability to bind more than two epitopes of the protein NGAL, (c) bringing into contact of the second sample fluid the body of the individual, where the sample obtained after the start of treatment, with the second device for analysis, including antibody to NGAL and detektiruya tag for the formation of a complex between NGAL protein in the second sample with the antibody to NGAL, (d) determining the amount of the second complex formed between NGAL second sample and antibody to NGAL in the second device for analysis, using a detectable label, where the antibody to NGAL in the device has the ability to associate more than two epitopes of the protein NGAL, and (e) comparing the amount of first complex number of the second complex, where the reduction amount of the second complex, compared to the amount of the first complex, indicates that the treatment is effective the M.

In an additional embodiment, the invention relates to a method for monitoring the effectiveness of treatment of acute kidney injury, where the method comprises the stage of: (a) bringing into contact of the first sample of body fluid of an individual, where the sample obtained prior to treatment with a polyclonal antibody to the associated with gelatinase of neutrophils to lipocalin (NGAL), (b) determining the amount of the first complex formed between NGAL protein in the first sample and a polyclonal antibody to NGAL using a detectable label, (c) bringing into contact the second sample of body fluid of an individual, where the sample obtained after the start of treatment with polyclonal antibody to NGAL, (d) determining the amount of the second complex formed between NGAL from the second sample and a polyclonal antibody to NGAL, using a detectable label, and (e) comparing the amount of the first complex formed between NGAL in the first sample and a polyclonal antibody to NGAL, with the amount of the second complex formed between NGAL from the second sample and a polyclonal antibody to NGAL, where the reduction amount of the second complex, compared to the amount of the first complex, indicates that the treatment is effective.

In yet another additional embodiment of the invention the invention relates to a set is to define acute kidney injury in the individual. In one of the embodiments the kit contains a device for analysis, comprising the antibody to the associated with gelatinase of neutrophils to lipocalin (NGAL) and detektiruya label, which is adapted for use in determining the amount of complex formed between NGAL in a sample of body fluid and antibody to NGAL, where the antibody to NGAL in the device has the ability to bind more than two epitopes of the protein NGAL

In another embodiment, the kit contains a first polyclonal antibody to associated with gelatinase of neutrophils to lipocalin (NGAL), adapted to interact with the sample of body fluid, the second antibody to NGAL, adapted for use in determining the amount of complex formed between the protein NGAL in a sample of body fluid and the first polyclonal antibody to NGAL, and detektiruya label, which is adapted for use in determining the amount of complex formed between NGAL in a sample of body fluid and the first polyclonal antibody to NGAL.

In an additional embodiment, the invention relates to a device for analysis to determine the acute kidney injury in an individual, where the device comprises a polyclonal antibody to NGAL, immobilized on the substrate, and adapted to interact with the sample of body fluid and d is tectorum tag adapted to associate with a complex of NGAL protein and immobilized polyclonal antibody to NGAL.

As methods, devices and kits according to the invention using the antibody to NGAL, with the ability to bind more than two epitopes of the protein NGAL, it is striking that the methods and kits show increased sensitivity to NGAL as a biomarker and thus exhibit increased sensitivity in the determination of acute kidney injury. Hypersensitivity can provide earlier identification of such damage and thus can afford to get an earlier response to treatment.

In an additional embodiment, the invention relates to a method of determining in a sample of individual protein neutrophil origin of lipocalin associated with gelatinase (NGAL). In a more specific embodiment, the methods can be used to distinguish NGAL renal origin and NGAL neutrophil. In one of specific embodiments of the invention, such methods include (a) determining the relative amounts of Monomeric, dimeric and heterodimeric forms of NGAL protein in the sample and (b) comparing the resulting definitions of quantities, where the overwhelming amount of Monomeric and/or heterodimeric protein NGAL compared to dimeric be the com NGAL, shows that NGAL protein is derived from the kidney of an individual, while equal or predominant amount of dimeric proteins NGAL compared to Monomeric or heterodimeric protein NGAL, demonstrates that NGAL protein is derived from neutrophils of the individual. Definition or clarification of the source of NGAL protein will facilitate the diagnosis of the condition and will allow, in particular, to spend more quality, targeted treatment.

These and other advantages and improvements will be better understood by considering the following detailed description.

Brief description of drawings

A detailed description will be more fully understood in light of the drawings, in which:

onfigure 1 shows the levels of creatinine in plasma before and after surgery, as described in example 1. The upper levels of the norm for men and women respectively 100 µmol/l and 90 µmol/L. Levels before surgery was significantly higher compared to normal levels for men and women (p<0,001).

On figa and 2B presents the levels of NGAL in the urine before and after surgery, measured by RIA analysis, using a polyclonal antibody and analysis, which uses two monoclonal antibodies, respectively, as described in example 1. The levels in healthy subjects are also presented. The horizontal line shows the upper 97,5 procenti the ü to healthy controls. The main difference between the levels before and after surgery was determined by ANOVA analysis and are presented in figures. For both analyses, the levels after surgery were significantly different from the levels prior to surgery in all three time points (p<0,001).

On figa-3C shows a box plot of the dependence between the levels of NGAL in the urine within 2 hours after surgery and the time of extracorporeal circulation time (ECC), where the levels measured by RIA analysis, using a polyclonal antibody by ELISA using polyclonal and monoclonal antibodies, and analysis, which uses two monoclonal antibodies, respectively, as described in example 1. Shows the statistical discrepancy and the fold increase in median.

On figa and 4B shows the dependence between urlname NGAL in the urine and GFR (cystatin C in plasma was measured using RIA analysis, which used a polyclonal antibody and analysis, which uses two monoclonal antibodies, respectively, as described in example 1. Presents the results of linear regression analysis.

Figure 5 presents the relationship between the quantitative measurement of NGAL protein in the urine with the use of RIA, which uses a polyclonal antibody to NGAL, and analysis, which uses two monoclonal antibodies, as described in example 1. L is Nany regression analysis: r 2=0,86, p<0,0001, n=331. The inset shows the dependence between the two analyses in the lower limit of concentration.

Figure 6 presents the results of the analysis Western blotting as described in example 2, various molecular forms of NGAL in the urine Obraztsov U1 and U2 obtained from two patients undergoing surgery on the heart.

Figure 7 presents the results of measurements of NGAL in the urine fractions after gel filtration on Superdex™-75, using analyses based on the different antibodies as described in example 2. The major molecular forms of NGAL in peak 1 and peak 2 are the dimer and monomer, respectively. The inset presents the results of amplification of peak 1.

On Fig presents the time dependence of the synthesis process NGAL cells HK-2, cultured in air-conditioned environment, defined in the specified time periods, as described in example 2. Values are presented as mean ± SD obtained from analyses of duplicate from three independent experiments. The marks * and ** correspond to p<0.05 and p<0.01 respectively.

On figa and 9B shows the levels of NGAL secreted by cells HK-2, grown, or in complete medium or complete medium, enriched with stimulating means (figa), or grown in serum-free medium for keratinocytes (K-SFM) or in K-SFM, enriched with stimulating means (IGV). Values are presented as mean ± SD obtained from analyses of duplicate from three independent experiments. Label *, ** and *** correspond to p<0.05 to p<0.01 and p<0,001, respectively.

Onfigure 10 on the bottom panel presents the determination of NGAL, which is secreted by cells HK-2) were cultured in an air-conditioned environment analysis, Western blotting, and the top panel presents the expression of NGAL mRNA cells HK-2, collected at the indicated time points after addition of fresh medium (C) or medium enriched with 1 ng/ml IL-β (S).

Various aspects, features and embodiments of the invention will be more fully understood in light of the detailed description.

Detailed description

Originally NGAL was isolated from human neutrophils and in previous papers it was shown that measuring the amount of NGAL in blood is an excellent method for detection of acute infections caused by bacteria and viruses. Later investigated the direct relationship between the excretion of NGAL, for example, in samples of body fluids such as urine, and acute kidney damage. Surprisingly, comparison of the results of measurement of NGAL through analysis of monoclonal antibody-monoclonal antibody and analysis using polyclonal antibody showed significant differences in clinical efficacy of these Ana is Izov. The results show that the choice of antibodies for analysis is crucial, and specifically the use of antibodies that can react with more than two epitopes of the protein NGAL, provide methods of analysis with high sensitivity, confirming that these methods of analysis identify different ways NGAL-apoptotic genes in different conditions. These methods, devices and kits use, thus the antibody to NGAL, which has the ability to interact with more than two epitopes of the protein NGAL. In this regard, such an antibody to NGAL may include one or more polyclonal antibody to NGAL, and/or combination of one or more polyclonal antibody to NGAL with one or more monoclonal antibody to NGAL as additionally described in more detail below. Additionally, the antibody against NGAL or antibodies can be used to capture protein NGAL, and/or can be used with a detectable label.

Methods for determining acute kidney injury in an individual can be used in any person, and specifically the individual, who may be at risk of developing acute kidney injury. Such individuals include, but are not limited to, individuals in the postoperative period after heart surgery or kidney transplantation, which was introduced on the agnostic means in vivofor example, a radiopaque contrast agent or nephrotoxic therapeutic agent and the like, and/or individuals with diabetes, septicemia, hemorrhagic shock or the like In a specific embodiment, the individual is a patient who had surgery on the heart and blood sample from the patient receive within three hours after surgery on the heart. In another embodiment, a method of determining repeat on the respective samples obtained through certain periods of time after surgery on the heart, for example 2 hours and 5 hours after surgery, after 2 hours and 12 hours after surgery, 2 hours, 12 hours and 24 hours after surgery or other

To determine ways to use the sample of body fluid. In a more specific embodiment, the sample comprises urine, blood, serum or plasma or purified component. In a more specific embodiment, the sample is urine.

In one of the embodiments the method comprises (a) bringing into contact a sample of body fluid of an individual with a device for analysis, including antibody to NGAL and detektiruya label that provides education complex NGAL protein in the sample with the antibody to NGAL, the (b) determining the amount of complex, formed between NGAL protein from the sample and the antibody to NGAL in the device for analysis using detektiruya label, where the antibody to NGAL in the device has the ability to bind more than two epitopes of the protein NGAL and where the amount of formed complex reflects the severity of acute kidney injury. As described above, the antibody to NGAL, with the ability to bind more than two epitopes of the protein NGAL can be provided by one or more different antibody to NGAL.

Qualitative or quantitative determination of the formed complex, which is an indicator of acute kidney injury can be calibrated to the particular device or method. In a specific embodiment, when the measurement is performed radioimmunological assay (RIA), the amount of NGAL protein that is an indicator of acute kidney injury is 60 ng/ml or more. In another embodiment, when the measurement is performed enzyme-linked immunosorbent assay (ELISA), the amount of NGAL protein that is an indicator of acute kidney injury is 100 ng/ml or more.

In one embodiment, the implementation of the methods using polyclonal antibody against NGAL, for example, as described in Xuet alJournal of Immunological Methods, 171:245-252 (1994), included in this the document is t as a reference. For example, as described in Xuet al, polyclonal antibody to NGAL (HNL) induce in rabbits by a large number of intradermal injection of purified protein, total number of 72 μg, homogenized in complete and incomplete Freund's adjuvant. The specificity of the antibodies can be assessed double immunodiffusion (Devereuxet al.,Nucleic Acid Research, 12(l):387-394 (1984)) agarose and tested against extracts of neutrophilic granules and the following purified proteins: cathepsin G, elastase, myeloperoxidase, lysozyme, lactoferrin, cationic protein of eosinophils (ECP) and protein X eosinophils (EPX/EDN). You can, of course, to use other antibody to NGAL.

In one of the embodiments of the methods according to the invention include (a) bringing into contact a sample of body fluid of an individual with a polyclonal antibody to NGAL and (b) determining the amount of complex formed between NGAL in the sample and a polyclonal antibody to NGAL, using a detectable label, where the number of complex reflects the severity of acute kidney injury. In a particular embodiment, for example, antibody to NGAL includes polyclonal antibody and the amount of complex formed between NGAL protein in the sample and the antibody to NGAL, define common ways radioimmunological analysis. Such methods are well known in the area and include, preferably, use of double radioimmunological analysis, where you can use two polyclonal antibodies or where you can use one polyclonal antibody and one monoclonal antibody. In another embodiment, the quantity of complex formed between NGAL protein in the sample and the antibody to NGAL, determine enzyme-linked immunosorbent assay (ELISA), where in ELISA using at least one polyclonal antibody to NGAL. The ELISA methods are also well known in this field. In specific embodiments, implementation, using the ELISA method, the inspection device includes a polyclonal antibody to NGAL monoclonal antibody to NGAL, where one of the antibody to NGAL is associated with the substrate, and the other antibody to NGAL is associated with a detectable label. In a more specific embodiment, polyclonal antibody against NGAL is associated, i.e. immobilized on the substrate. In an additional embodiment, the polyclonal antibody to NGAL is associated with the substrate, and a monoclonal antibody to NGAL is associated with a detectable label. Alternative to ELISA, you can use the device for analysis, including two different polyclonal antibody against NGAL, one such antibody to NGAL is associated with the substrate, and the other is associated with a detectable label. You can use is to use other known in the field of methods of analysis, where, for example, at least one polyclonal antibody to NGAL immobilized on the substrate, and in more specific embodiments, the implementation of the detected label is associated with a different antibody to NGAL, which can be a monoclonal antibody to NGAL, and a second polyclonal antibody to NGAL.

Thus, the invention also relates to devices and kits for such methods. In one of the embodiments of the device for analysis according to the invention includes polyclonal antibody to NGAL, immobilized on the substrate and adapted for interaction with a sample of body fluid, and the detected label is adapted to associate with a complex of NGAL protein and immobilized polyclonal antibody to NGAL. Detected mark may, in particular embodiments, implementation, exist in complex with antibody to NGAL or monoclonal, or polyclonal for binding NGAL protein that forms a complex with immobilized polyclonal antibody to NGAL. The inspection device may be provided as a device or set to “diagnostics”, which facilitates its use by medical personnel.

As will be clear that the invention can additionally be used to control the treatment of acute kidney injury by analyzing large number of samples indie is iguana before and after or during a treatment regimen. Such methods generally include the bringing into contact of the first sample of body fluid of an individual with the first device for analysis, as described, and determining the amount of complex formed between NGAL protein in the first sample and the antibody to NGAL in the first device for analysis, bringing into contact the second sample of body fluid of an individual, where the sample obtained after the start of treatment, with the second device for analysis, as described, and determining the amount of the second complex formed between NGAL from the second sample and the antibody to NGAL in the second device for analysis, and the comparison of the first complex number the second complex. The reduction in the number of the second complex compared to the amount of the first complex shows that the treatment is effective.

In another embodiment, the invention relates to methods for determining the origin of lipocalin, a protein associated with gelatinase neutrophils (NGAL) in a sample of the individual. Such methods are especially useful for distinguishing between protein kidney NGAL and NGAL protein of neutrophils. In one of the embodiments the methods include (a) determining the relative amounts of Monomeric, dimeric and heterodimeric forms of NGAL protein in the sample and (b) the comparison result to determine which of richest, where the overwhelming amount of Monomeric and/or heterodimeric protein NGAL compared to the dimeric protein NGAL, shows that NGAL protein is derived from the kidney of an individual, while equal or predominant amount of dimeric forms of NGAL protein in comparison with Monomeric or heterodimeric form of NGAL protein, shows that NGAL is derived from neutrophils of the individual. It is shown that NGAL protein renal origin essentially does not contain dimeric form of the protein NGAL, for example, as shown by analysis of Western blotting. In a specific embodiment, the sample contains urine. In another specific embodiment, the appropriate amount of NGAL protein in the sample is determined by bringing into contact of the sample with a device for analysis, including monoclonal antibody to NGAL. In another embodiment, the appropriate amount of NGAL protein in the sample are determined by the interaction of the sample with a device for analysis, including polyclonal antibody to NGAL. You can use any device for the analysis and methods, such as described above, Western blotting, or other conventional methods and/or devices.

As shown in example 2 below, polyclonal and monoclonal antibodies recognize (i.e. form a complex with them) Monomeric, dimeric and heterodimeric forms of NGAL protein. However, for white is and NGAL renal origin in the interaction essentially not detected dimeric form of the protein NGAL, while the dimeric form of NGAL are predominant in the results obtained for the NGAL protein from neutrophils. Not wanting to be limited by theory, believe that the different types of protein NGAL exhibit different epitopes, which are then linked, in various ways, for example, monoclonal antibodies. Thus, the comparison, as described, may allow to distinguish between NGAL protein renal origin from protein NGAL neutrophil origin.

Various aspects of the invention are illustrated in the following examples.

EXAMPLE 1

This example describes a study of the determination of NGAL using antibody to NGAL, with the ability to bind more than two epitopes NGAL protein, and comparing the received data with the determination of NGAL, using two monoclonal antibodies.

Patients and samples

In the study ofincluded a total of 59 adult patients who underwent surgery in the heart of Uppsala University Hospital. The age of the patients varied in the range 27-85, on average, 63 and in the number of patients included 42 men and 17 women. Surgery on the heart included 23 coronary artery bypass surgery, 15 operations prosthetic aortic valve, 4 operation to restore the function of the mitral valve, 3 combined operations 8 operations and the plantations of assistive devices in the left ventricle and 6 other operations.

Samples of urine and blood were collected before surgery and at different time points (2, 24, 48 and 72 hours) after surgery on the heart. Urine samples immediately centrifuged at 3,000 rpm./min at 4°C for 15 min Plasma with EDTA was obtained by centrifugation of blood at 3,000 rpm./min at 4°C for 15 minutes All samples supernatant was immediately divided into aliquots and kept at -20°C. Additional samples of urine number 101 was collected from healthy employees and students, and they served as normal controls.

Analyses of the levels of NGAL in the urine

The levels of NGAL was measured in three different analyses. In the first method of analysis used RIA, based on polyclonal antibodies, in accordance, generally, methods Xuet alabove. The second method of analysis used ELISA based on monoclonal-polyclonal antibodies. In the third method of analysis used analysis based on monoclonal-monoclonal antibodies. Thus, the first two methods are presented according to the invention, whereas the third method used to compare.

More specifically, did radioimmunological assay (RIA) based on polyclonal antibodies, as described previously Xuet alwith some modifications. 50 μl of a solution of each sample or standard (2 µg/l to 128 mg/l) were sequentially mixed is with 50 μl of NGAL, labeled I125and 50 µl of the specific antibody, appropriately diluted in buffer for RIA analysis. The mixture is incubated at room temperature for 3 hours. Next was added 500 μl of the suspension adsorbed on the cellulose solid-phase secondary antibodies (AA-SAC1, IDS LTD, England) and incubated for 1 hour at 4°C. the Complexes of antibody to NGAL associated adsorbed on cellulose antibody to rabbit IgG was separated and precipitated by centrifugation at 3400 rpm./min for 15 minutes. After desantirovaniya measured radioactivity. The coefficients of variation (CV) analysis and a series of tests was less than 6% and 10% respectively. The results obtained when measuring the concentration of NGAL in the urine through the device for the analysis of RIA, identified as NGAL RIA.

In this study, has developed a device ELISA based on polyclonal and monoclonal antibodies. In brief, tablets for micrometrology (Nunc Maxsorp, Agogent, Denmark) were coated with monoclonal antibody to NGAL (100 μl/well, 1 μg/ml)diluted in carbonate-bicarbonate buffer (0,05M Na2CO3-NaHCO3pH of 9.6, Invitrogen Corporation, UK) at 4°C. Additional binding sites blocked in carbonate-bicarbonate buffer containing 2% bovine serum albumin (200 μg/well, Sigma-Aldrich, Steinhein, Germany) at 37°C for 1 hour. 100 μl of standard samples (0.1 ng/ml is about to 6.4 ng/ml) or diluted samples, dissolved in solution for analysis (PBS containing 0.2% bovine serum albumin, 0.1% of Tween-20, 0.05% of CTAB and 0.02% NaN3), was added with duplicate and incubated at room temperature for 2 hours. Then per well was added 100 μl of the diluted polyclonal rabbit antibodies to NGAL, and incubated at room temperature for 1 hour, followed by addition of 100 μl of the diluted antibodies conjugated to horseradish peroxidase (GE Healthcare, UK) and incubated at room temperature for another hour. The enzymatic reaction on the plates were visualized with a solution of 3,3',5,5'-tetramethylbenzidine (100 µl/well, Sigma-Aldrich, Steinhein, Germany) at room temperature for 20 min and stopped by adding 1M H2SO4at the rate of 100 µl/well. The tablets were washed four times in buffer for washing (PBS containing 0.05% Tween-20) between each of these stages, using a Microplate Washer (Anthos fluido, Salzburg, Austria). The optical density was measured at 450 nm with correction for readings at 540 nm in the control holes through microspectrophotometry read tablets (SPECTRAmax 250, GMI, Inc., USA). The average value of CV analysis was 2.8% (range from 0.5 to 4.7%), and CV for a series of analyses was 6.3 (range from 2.1 to 10.4%). The average extraction rate was 99% (range from 93 to 105%). The results obtained by measuring the concentration of NGAL in the urine by ELISA method, refer to the to NGAL ELISA.

Analysis of NGAL double monoclonal analysis was performed according to the manufacturer's instructions. The coefficients of variation analysis and a series of tests (% CV) was less than 6%. The results of measuring the concentration of NGAL in the urine measured by this device, known as NGAL mono-mono.

The levels of creatinine in urine was measured on the device Architect accepted way in the Department of clinical chemistry at the University hospital in Uppsala and used to adjust the levels of NGAL in the urine due to variations in dilutions of urine. Thus, the levels of NGAL in the urine represented as NGAL in µg/mmol creatinine. All measurements were performed in duplicate and researchers in the laboratory until the end of the study did not know about the origins of the samples and clinical outcomes.

Western blotting NGAL in urine

Western blotting was performed as described previously Towbinet al,Proc. Natl. Acad. Sci.USA, 76:4350-4 (1979). In brief, 20 µl of urine sample was placed in the gel Nu-PAGE® 4-12% Bis-Tris (Invitrogen Corporation, USA). After SDS-PAGE, proteins were transferred to PVDF membrane using the clipboard to transfer Nu-PAGE® (Invitrogen Corporation, USA) at 25 V for 1 hour. Additional binding sites on the PVDF membrane was blocked with a solution to block (GE Healthcare, UK) for 1 hour. The blots were incubated with monoclonal mouse antibodies to NGAL for 1 hour followed by incubation for 45 min with in origname antibodies, conjugated with horseradish peroxidase (GE Healthcare, UK). Immunoblot were detected using enhanced chemiluminescence according to the manufacturer's instructions (Amersham ECL™ Western Blotting System, GE Healthcare, UK).

Additional tests

The levels of creatinine and cystatin-C in plasma was measured using conventional methods, in the Department of clinical chemistry University hospital in Uppsala.

Statistical analysis

Non-parametric tests Mann-Whitney and Wilcoxon for unpaired and paired comparisons, linear regression analysis, univariate analysis of variance (ANOVA) was performed Medcalc 9,5 (MedCalc Software, Mariakerke, Belgium) and STATISTICA to 8.0 (StatSoft, Inc., Tulsa, USA). Statistical significance was p<0,05.

Results

The creatinine levels in plasma before the surgery, and up to 78 hours after surgery are presented in figure 1, not shown differences between the results in the presented periods of time. Clinical outcome showed that three of the subjects showed signs of acute renal impairment with creatinine levels in plasma after surgery >50%.

The levels of NGAL in the urine

The levels of NGAL in the urine obtained from healthy subjects and in patients who have undergone surgery on the heart, measured using the three described methods. The determination results, obtained using the receiving ways RIA and mono-mono, presented respectively on figa and 2B. Levels before the surgery were comparable to levels in normal controls. Two hours after surgery levels significantly increased (p<0.0001)and approximately half of the patients have readings above the upper limit of normal controls. The values of the median, in the case of measurement by RIA method, represented an increase of 18.7 15.6 and 11.4 times in the case of measurement by ELISA method and by way of mono-mono, respectively. After 24 hours the values of the levels again declined, but their value was still higher than the values of the levels before the surgery. The magnitude of the levels remained significantly higher during the postoperative period (p<0,0001). In 72 hours the rate of increase amounted to 6,8 respectively, of 8.5 and 5.9 for ways RIA, ELISA and mono-mono. A similar pattern was observed for all three methods during the whole time.

The relationship over time, artificial circulation (ECC)

Found a significant positive correlation between ECC and values of NGAL levels obtained 2 hours after the operation, when measured in urine by RIA analysis (r2=0,30, p<0,0001) and ELISA (r2=0,16, p=0.006). However, for the analysis of mono-mono, this correlation is not found. When divided into subgroups according to the time ECC in excess of or less than 90 mi is, the results of the RIA is increased by 12.6 times in samples obtained 2 hours after surgery (p=0.006). The results of the ELISA was increased 6.5 times (p=0,027) and the results of the analysis of mono-mono 5.2 times (p=0.07), as shown in figa-3C.

The relationship between the levels of NGAL in the urine and kidney function

In plasma as indicators of kidney function measured creatinine levels and cystatin-C. As shown above, the values of creatinine remained unchanged in the majority of the subjects, only three of the subject after the surgery had evidence of acute renal impairment, characterized by increased levels of >50%. Value levels cystatin-used to calculate the level of glomerular filtration rate (GFR). In univariate analysis, GFR was associated with NGAL (RIA) (r2=0,28, p<0.001) and NGAL (mono-mono) (r2=0,25, p<0,001), as shown in figa and 4B. Also analyzed the correlation between the level of NGAL in the urine and creatinine levels in plasma. Subjects were divided into two groups according to the percentage increase in creatinine in the plasma within 72 hours of the postoperative period compared with the baseline (<120% or >119%). The value of NGAL levels (RIA) within 2 hours after surgery were significantly higher in the group with increased levels of creatinine >119% (p=0.03), in contrast to values of NGAL levels (mono-mono), which were not significantly increased (results not presented).

The correlation between the three methods of analysis of NGAL

The overall correlation between NGAL (RIA) and NGAL (mono-mono) is shown in figure 5 (r2=0,86, p<0,0001, n=331). Correlations at different points in time are presented in table 1, and shows a very good correlation values with r2located between 0,952-0,996, but with the exception of the results obtained within 2 hours after surgery. At this point in time, r2was 0,680 and was significantly lower than others (p<0,0001). The relationship between NGAL (RIA) and NGAL (mono-mono) in the group of healthy in appearance entities amounted to r2=0,887 and also significantly different from the results obtained after 2 hours (p=0.001). Regression analysison Passing and Bablok all 331 results led to the equation HNL (RIA)=0,6553+0,5358×NGAL (mono-mono) with a significant deviation from a straight line (p<0,01), which also occurred in the case of a comparison of methods for the analysis of NGAL (mono-mono) and NGAL (ELISA), NGAL (ELISA)=0,0370+0,1135×NGAL (mono-mono). However, comparison of methods for the analysis of NGAL (RIA) and NGAL (ELISA) led to the equation NGAL (ELISA)=-0,002192+0,2002×NGAL (RIA) without deviating from the straight line.

Molecular forms of NGAL in the urine

The prevailing forms of NGAL detected in the urine before and after surgery on the heart, had a relative molecular mass of 25 (monomer), 45 (glycosilated) and 90-130 kDa (complexes with MMP-9), respectively. Representing the th of these various forms varied before and after surgery. Investigated the relationship between homodimers and monomers, based on the scanning of the Western blot. It is shown that the relative number of homodimers increased up to 24 hours after surgery (p=0.02), after which the ratio tended to decrease.

Discussion

The results presented herein showed that the choice of antibodies in the way of analysis of NGAL is fundamental to identify the various options NGAL secreted into the urine under different conditions. Specifically, the methods of analysis using antibody to NGAL, which are able to react with more than two epitopes of the protein NGAL, provide enhanced sensitivity.

This study included adult patients undergoing surgery on the heart. Acute kidney injury is one of the most serious postoperative complications that can affect these patients. In this study, mean values of creatinine in plasma remained unchanged, only three of the subjects had an increase of >50% as sign of acute kidney injury. Despite this, the increase in 10-100 times the level of NGAL in the urine found in approximately half of patients, only 2 hours after the operation. In addition, NGAL levels remained unchanged the mi during the whole observation period. In General, the value of NGAL levels showed a weak but significant correlation with renal function, as shown by measurement of the levels of cystatin C or creatinine in the plasma, which supports the assumption that NGAL is an earlier and more sensitive marker of renal dysfunction. Moreover, two of the three patients with elevated creatinine >50% were observed significantly elevated levels of NGAL in 2 hours after surgery. From this study it is evident that a significant increase in the level of NGAL was in the early period after surgery, half of the patients, but that this increase was only an intermediate, followed by a gradual increase in the coming days for all patients. Thus, without wishing to be limited by theory, these results may reflect different mechanisms of the processes included in the excretion in urine NGAL. Early phase may reflect the excretion preformed NGAL from various sources, such as the epithelium of the kidneys and populations of neutrophils, whereas later excretion may reflect a synthesis in the kidneysde novo. Results differences in the extent to which presents in the urine a lot of different size molecules NGAL in different periods of time, also suggest the involvement of different mechanisms of the process.

Comparison of three different methods of analysis showed snickersnee. In General, the methods of analysis were significantly correlated, with some obvious exceptions. These exceptions were the most represented in the samples obtained 2 hours after the operation, as a confirmation of the fact that three of the tests in these conditions measure different molecular variants of NGAL. These differences further exemplify the fact that the results of the analysis of RIA and ELISA, using polyclonal antibodies, compared with the analysis of mono-mono, showed a close relationship with clinical parameters such as the length of time of extracorporeal circulation and kidney function. These results thus showed that the identification of all forms of NGAL in the urine is important for clinical characteristics analysis. RIA is an analysis based on polyclonal antibodies, which probably sent to all epitopes on the molecule, whereas the analysis based on monoclonal-monoclonal antibodies, directed only on the identified epitopes, some of which may or may not be masked by the formation of complexes or other molecular interactions. The ELISA based on polyclonal-monoclonal antibodies, demonstrated settings, in some degree intermediate between these two extreme cases, which can be explained facto is, this analysis allows us to recognize more epitopes than double monoclonal analysis, but less than the analysis based on polyclonal antibodies. In essence, these results confirm that NGAL is a useful early biomarker of postoperative renal impairment, when it is measured in the urine and demonstrate again that the configuration of the antibodies in the analysis has an impact on the clinical characteristics of the analysis, since some forms of NGAL may not be detected by tests that use antibodies with limited specificity.

EXAMPLE 2

In this example, describe the study definition of NGAL in respect of Monomeric, dimeric and heterodimeric forms when determining the source of NGAL protein.

Urine samples and separation by gel-filtration

Overall, 33 of the urine sample was collected before surgery and at time 2 h and 24 h after surgery on the heart. Urine samples immediately upon receipt was centrifuged at 3,000 rpm./min for 15 min at 4°C and stored divided in aliquots at -20°C. Gel filtration of a single urine sample taken after 2 h after the operation was performed on the filled columns Superdex™ 75 HR 10/30 using the FPLC system (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Fractions of 250 l was collected and stored at -20°C. Buffer for elution predstavlyalsya PBS. NGAL in the fractions was determined using RIA and ELISA, as described below.

The sensitivity of ELISA for the qualitative determination of NGAL

Used six ELISA based on different antibodies for the quantitative determination of NGAL, namely 1) Mab697-polyclonal (monoclonal-polyclonal ELISA as described in example 1), 2) Mab764-Mab765, 3) Mab764-polyclonal, 4) polyclonal-Mab765, 5) polyclonal-polyclonal and Mab-697-Mab765. Basic protocols for these five ELISA was the same as described in example 1, except for the specific antibodies used in the analysis. In brief, 96-well plates to micrometrology (Nunc Maxsorp, Agogent, Danmark) covered polyclonal antibody rabbit or monoclonal antibody mouse (Mab697 and Mab764) to NGAL% human. (Diagnostics Development, Uppsala, Sweden). Samples and standards (in the range 0,039-5 µg/l) (100 µl/well) were incubated for 60 min (urine samples and fractions after gel filtration) or 90 min (supernatant cell culture) at room temperature (RT). Then 100 μl/well of the diluted biotinylated polyclonal rabbit antibodies or monoclonal antibody mouse (Mab765) to NGAL people were added and incubated at RT for 60 min, followed by addition of 100 ál/well of diluted streptavidin conjugated to horseradish peroxidase (GE Healthcare, United Kingdom) (30 min at RT). Plates were washed Chet is d times in the buffer for washing (PBS, containing 0.05% Tween-20)using a Microplate Washer (Anthos fluido, Salzburg, Austria) between all stages. The enzymatic reaction was visualized by means of a solution of 3,3',5,5'-tetramethylbenzidine (100 µl/well) (Sigma-Aldrich, Steinhein, Germany) as substrate at RT for 15 min and the reaction was stopped by adding 1 M H2SO4(100 µl/well). The optical density was observed at 450 nm on a spectrophotometer (SPECTRAmax 250, GMI, Inc., USA.

RIA, based on polyclonal antibodies for the quantitative determination of NGAL

RIA was performed as described above. In brief, 50 µl of sample or 125 controls (2 ug/l and 128 ug/l) was mixed with 50 μl of NGAL, labeled I125and 50 µl of specific antibodies. The mixture is incubated for 3 h at room temperature. Next, 500 μl of the suspension adsorbed on cellulose secondary antibodies (AA-SAC1, IDSLTD, United Kingdom) was added and incubated for 1 h at 4°C. the Complexes NGAL antibodies associated with adsorbed on cellulose antibodies to rabbit IgG, besieged by centrifugation. After decanting measured radioactivity.

Culture HK-2 and expression of NGAL protein

HK-2 (human kidney 2, CRL-2190) taken from the American type culture collection (ATCC). This cell line epithelium of the proximal tubules of the kidneys obtained from kidneys without pathology. Cells were subjected to the immortalization by transduction of the genes E6/E7 of human papilloma virus 16 (HPV-16). The granulosa cells or in the culture medium (serum-free medium for keratinocytes (K-SFM), enriched with 0.05 mg/ml of the extract of the pituitary gland of the ox (BPE) and 5 ng/ml human recombinant epidermal growth factor (EGF) (Invitrogen-Gibco®, United Kingdom), or in incomplete medium for cultivation at 37°C in a humid atmosphere with 5% CO2. Additionally, cells were cultured with specific stimulants, including cytokines (IL-1β or TNF-α (Sigma-Aldrich, Steinhein, Germany) and LPS (Invitrogen-Giboco®, United Kingdom). 0,5×105cells and 1 ml of complete medium for cultivation per well were cultivated in 24-well plates (FALCON®, USA). After 48 h subculturing complete culture medium was removed and the monolayer (approximately 90% confluence) were twice washed in PBS (Invitrogen-Giboco®, United Kingdom). The cells were maintained in air-conditioned environment during the period of time of 72 hours Supernatant medium was collected for the quantitative determination of NGAL in 2 h, 12 h, 24 h, 48 h and 72 h, respectively.

Evaluation of NGAL gene expression RT-PCR

Cultivated in the usual way and induced by 1 ng/ml IL-1β cells HK-2 were collected at 2 h, 4 h, 6 h, 8 h, 12 h and 24 h for isolation of total RNA. RNA was isolated using the RNeasy kit® Mini (QIAGEN, United Kingdom) according to the manufacturer's Protocol. Single-stranded cDNA was synthesized using reverse transcriptase SuperScript III (Invitrogen, United Kingdom), 200 ng of total RNA. The polymerase is th chain reaction (PCR) was carried out, using DNA polymerase Taq (Invitrogen, United Kingdom) in a DNA Engine PCR machine (PTC-200) (Bio-Rad, USA). Sequence-specific oligonucleotide primers for NGAL (5'-TCACCTCCGTCCTGTTTAGC-3' and 5'-CGAAGTCAGCTCCTTGGTTC-3') and β-actin (5'-TTCTACAATGAGCTGCGTGTGG-3' and 5'-GTGTTGAAGGTCTCAAACATGAT-3') were chosen according to literature data and synthesized by Thermo SCIENTIFIC (Germany). Initial denaturation condition consisted of heating at 94°C for 2 min. Amplification by PCR was performed using the stage of denaturation for 30 sec at 94°C, followed by a stage of annealing for 30 sec at 60°C (NGAL) or 59°C (β-actin) and the stage of elongation for 30 sec at 72°C. In General, for both genes, the reaction was carried out for 30 cycles, followed by final elongation for 10 min at 72°C. PCR Products were separated by electrophoresis in 2% agarose gel and was determined by staining with methyl-ethidium. The expected size of PCR products (242 BP and 119 BP for NGAL and β-actin, respectively) verify towards the stairs DNA increments of 50 BP (DirectLoad™ DNA Marker (Sigma-Aldrich, Steinhein, Germany).

Western blot

Productsreleaseneutrophilic granules was received as described. Collected supernatant HK-2 air-conditioned environment at time 72 h and was added to 0.1 mm PMSF (Sigma-Aldrich, Steinhein, Germany) and pills containing a set of protease inhibitors Complete™ (Roche, Mannheim, Germany). Supernatant con who was entrirely, using a device with a filter centrifuge Amicon® Ultra-4 (10,000 MW) (Millipore, USA). SDS-PAGE and Western blotting was performed according to the manufacturer's instructions. In brief, 25 μl or urine or concentrated supernatants air-conditioned environment or product release of neutrophilic granules were applied to the gel 4-12% Bis-Tris Nu-PAGE® (Invitrogen, USA) under conditions not conducive to recovery. Proteins were transferred to PVDF membrane Hybone-P (GE Healthcare, United Kingdom)using the clipboard to transfer Nu-PAGE® (Invitrogen, USA) at 25V for 1 hour. Additional binding sites on the PVDF membrane was blocked with a solution to block (GE Healthcare, United Kingdom) for 1 h, Blots were incubated overnight at RT or with polyclonal rabbit antibodies, or monoclonal mouse antibodies (Mab 697, Mab 699, Mab 763, Mab 764 or Mab 765) or a mixture of monoclonal antibodies to human NGAL, followed by incubation for 1 h with secondary antibodies conjugated with horseradish peroxidase (GE Healthcare, United Kingdom). Immunoblot was determined using enhanced chemiluminescence according to the manufacturer's instructions (Amersham ECL™ Western Blotting System, GE Healthcare, United Kingdom).

Statistical analysis

Student test and univariate analysis of variance (ANOVA) was performed by means of STATISTICA to 8.0 (StatSoft, Inc., Tulsa, USA) and Medcalc 9,5 (MedCalc Software, Mariakerke, Belgium). Values are presented as mean ± SD and median with mezhkvartalny what asmahan. p<0.05 is considered as significant.

Results

Determination of the molecular forms of NGAL in the urine analysis Western blotting

One kind of polyclonal antibodies rabbit and five types of monoclonal antibodies mouse to NGAL was used for the identification of the molecular forms of NGAL presented in the urine, which was obtained from patients subjected to surgery on the heart. In experiments on Biacore tested five monoclonal antibodies on the ability to react with different epitopes. As shown in Fig.6 for two typical samples of urine (U1 and U2), found significant differences between the characteristics of the antibodies. Three main bands are systematically identified by polyclonal antibodies and identified as Monomeric and dimeric forms of NGAL and forming complexes, heterodimeric forms of NGAL. These three forms were also determined by Mab764 and 765. However, with each antibody was additionally observed the extra band. However, it seems that the polyclonal antibody has a stronger affinity for dimer and a lower affinity to heterodimer than two monoclonal antibodies. Mab764 and 765 had very similar characteristics, by definition, all three molecular forms. Mab764 and Mab765 to NGAL had an affinity, from high to low, with Monomeric, heterodimeric and dimeric forms according to the respectively. Also it is shown that Mab763, and 697 699 had a high affinity to heterodimeric forms, whereas the affinity for the dimeric and Monomeric forms was weak. However, the ability of polyclonal antibodies and Mab765 and 697 to the determination of Monomeric and dimeric forms in supernatant stimulated neutrophils were very similar.

Characteristics of RIA and five ways ELISA for measuring NGAL in urine

Performance RIA and five ways ELISA are presented in table 1.

Table 1
Measurement HNL/NGAL in the urine samples obtained from patients undergoing surgery on the heart, through various methods of analysis
AnalysisBefore surgery, ug/lAfter 2 h after surgery, ug/lAt 24 h after surgeryFold increase (before surgery/2 h after surgery)Student test, p valueANOVA
a value of p
RIA7,19
(2,9-20,3)
248,20
(109-316,1)
26,96
(16,3-50,71)
34,5 0,0000110,0000020
ELISA 1
(Mab697-polyclonal)
0,94
(0,15-3,13)
28,82
(23,32-37,96)
4,75
(2,59-9,89)
30,70,000350,00027
ELISA 2
(Mab764-Mab765)
6,22
(1,16-12,8)
192,80
(78,2-287)
15,50
(of 9.55 40.7 in)
31,00,0000550,00002
ELISA 3
(Mab764-polyclonal)
is 3.08
(1,08-10,81)
239,10
(61,6-296,40)
19,80
(6,25-55,35)
77,60,0000530,00015
ELISA 4
(polyclonal-Mab765)
of 2.26
(0,79-7,84)
164,10
(45,9-207,1)
of 13.05
(of 5.89-40,2)
72,60,000100,000094
ELISA 5
(Polyclonal-polyclonal)
2,96
(1,13-13,68)
220,00
(58,4-249,9)
19,00 (7,5-58,15)74,30,0000240,000079

ELISA 6
(Mab697-Mab765)
1,27
(0,32-3.46 in)
30,00
(6,4-30)
of 3.46
(2,07-10,35)
23,60,000990,00012
Values are presented as median and interquartile range. Student test was performed between groups preoperatively and groups after 2 h after surgery and ANOVA was performed among the groups before surgery and groups after 2 h and 24 h after the operation.

Measured the levels of NGAL in the urine samples obtained before surgery, and in samples obtained after 2 h and 24 h after surgery, and the value of the median levels for NGAL, obtained through the analyses presented in table 1. The levels of median NGAL obtained in samples taken before surgery and 2 h after surgery, measured by the RIA method was the highest among the seven methods of analysis. On the other hand, the magnitude of the levels obtained ELISA based on Mab697 (1 ELISA and ELISA 6)was significantly lower compared with other analyses. Table 1 presents the differences in NGAL levels before surgery and 2 h after surgery, the differences, in General, during a 24-hour period. For all methods of analysis shows highly significant differences before surgery and after surgery. Multiple zoom levels, median before surgery 2 h after surgery was the highest and >70 in the case of measurement by ELISA method 3 (Mab764-polyclonal), ELISA 5 (polyclonal-polyclonal) or ELISA 4 (polyclonal-Mab765) and 23-34 times in the case of measurement RIA, ELISA 2 (Mab764-Mab765), 1 ELISA (Mab 697-polyclonal) or ELISA 6 (Mab697-Mab765).

Measuring NGAL in different ways analysis after gel filtration of the urine samples

Based on the results of the analysis Western blotting following two series of experiments were undertaken to study the characteristics of the methods of analysis to determine the various forms of NGAL. Performed gel filtration of a single urine sample obtained after 2 h after the operation on the column SuperdexTM 75 HR. The levels of NGAL in the fractions was measured by RIA and five ways ELISA and presented on Fig.7. Through the five ways ELISA received two peaks, corresponding eluting amounts of Monomeric and dimeric forms, respectively, and only one peak by RIA method. The latter is due probably to lack of sensitivity of the RIA method. The most significant levels of NGAL in peak 2 was obtained by RIA method and the lowest levels way 1 ELISA (ELISA-based Mab697-polyclonal). By all means the ELISA analysis, except ELISA 1, identified similar levels in peak 1, i.e. NGAL dimer (figure 2, insert). Analysis of ELISA 1 measured higher levels of NGAL dimer.

NGAL is activated in cells HK-2, when cells are growing under stressful conditions

Cells HK-2 were cultured for various periods of time in serum-free medium for keratinocytes (K-SFM), K-SFM enriched, or 0.05 mg/ml of the extract of the pituitary gland protein (BPE), or 5 ng/ml recombinant epidermal growth factor, human (EGF), or in complete culture medium recommended by ATCC (K-SFM, enriched with 0.05 mg/ml BPE and 5 ng/ml EGF), followed by cultivation for 48 h under standard conditions. The levels of NGAL in supernatant culture was determined at different time points (2 h, 12 h, 24 h, 48 h and 72 h) for 72 h period by ELISA 4. After 12-72 hours of cultivation the levels of NGAL in supernatant culture with K-SFM were significantly higher than in the other three environments for cultivation (Fig). The lowest levels were found in cells that were grown in complete culture medium. The results also suggest high levels of NGAL in supernatant cells, which were grown in K-SFM, enriched BPE, compared with cells that were grown in K-SFM, enriched rEGF. Overall, these results demonstrate the activation products NGAL in stressful conditions, in which cells are deprived of necessary factor is in growth.

NGAL is activated in cells HK-2 through IL-β, LPS and TNF-α

Cells HK-2 were grown in complete culture medium for 48 h, after which cells were additionally raised during various time periods in the presence of complete medium for the cultivation of enriched or IL-β (1 ng/ml, human), LPS (125 ng/ml,Klebsiella pneumoniae), or TNF-α (20 ng/ml, human). As shown in figa, IL-β induced the most significant increase of NGAL levels in the supernatant (increase from 8.9 to 41.9%). Also incubation with TNF-α and LPS induced a slight increase of NGAL levels in supernatant (2.2 and 1.6 times, respectively), but less significant than in the case of IL-β (p<0,001). Cells HK-2 were cultured in K-SFM, enriched IL-β, TNF-α or LPS. Significant increase NGAL has been shown to IL-β (increase from 1.3 to 12.8 times), but not with TNF-α or LPS (pigv). However, compared to cells grown in complete medium for cultivation, this increase was statistically less significant (p<0,001).

Molecular forms of NGAL produced by cells HK-2

Molecular forms of NGAL, secreted by cells HK-2 was determined by analysis of Western blotting using mixed monoclonal antibodies (Mab697, Mab764 and Mab765) as the detecting antibody. The results presented in figure 10 (bottom panel)show that the basic form of NGAL, which is secreted by cells HK-2, growing and or in complete medium for cultivation, or in a stressful environment, K-SFM, or in an environment enriched with cytokines (IL-β or TNF-α or LPS, is Monomeric form. Also after stimulation with IL-β was observed heterodimeric form of NGAL, whereas the dimeric form is not unlike the results obtained in supernatant human neutrophils (6). Figure 10 presents the levels of NGAL mRNA to cells HK-2 after incubation with IL-β. The results showed increased expression, which indicates that the active synthesis of NGAL cells HK-2.

Discussion

Originally NGAL was isolated from human neutrophils, and the authors have previously shown that the measurement of NGAL in blood is an excellent way to detect the presence of acute infectious diseases caused by bacteria or viruses. Further studies have shown that NGAL, under certain conditions, can also be expressed in other cells such as kidney cells, liver cells and epithelial tissue, and that the measurement of NGAL in the urine and plasma may serve as a biomarker of acute kidney injury. Example 1 shows that the configuration of the antibodies in the analysis of NGAL has influence on the clinical characteristics of the analysis. Some forms of NGAL was detected in the urine of patients with AKI. This example additionally shows that the Monomeric form and in some degree heterodimeric forms are the predominant FD is Mami, which are produced by canalave epithelial cells, while the dimeric form, apparently, is unique for neutrophils (see Fig.6). Neutrophils also produce Monomeric form. One interesting result of this study is the difference in the recognition of these various forms used by the antibodies, given that Monomeric and demarie forms originating from neutrophils, identified all monoclonal antibodies and polyclonal antibodies. These data disagree with the fact that Mab697 almost completely unable to recognize these forms in the urine. Also Mab765 showed strong reactivity against these forms in supernatant neutrophils, but only a weak ability to recognize dimeric form in the urine. Not wishing to be bound by theory, suggest the presence of differences in the epitopes, which exhibited various forms of NGAL, and, thus, are the cause of differences in molecular structures.

A significant difference in determining the amount of NGAL in the urine by means of methods of analysis also indicates the presence of different molecular forms of NGAL in the urine and differences in the recognition of epitopes by antibodies. Very different not only the levels before the operation, despite the same calibration device used in the analysis of the x, but also the relative change levels after surgery. Obviously, multiples increase was the largest value when measured by ELISA, using only or polyclonal antibodies, or polyclonal antibodies or in combination with Mab764, or Mab765. These two mab represented antibodies that also recognize the greatest number of forms in the urine analysis Western blotting. However, the combination of these two mab had less capacity to recognize, which means that additional molecular forms linked polyclonal antibody. From experiments with gel-filtration can be assumed that differences in the recognition of various forms primarily associated with differences in the recognition of a Monomeric forms of NGAL, because it seems that only one method of analysis allows for the detection, otherwise, dimeric form. Differences can not be explained by the analytical characteristics of the methods of analysis in General, because all methods of analysis showed the same sensitivity, accuracy, recovery, etc.

Based on the fractional excretion of NGAL in humans (CNGAL/CCr),in situhybridization in mice and the fact that NGAL is an acute phase protein, in previous reports described that the accumulation of NGAL in the urine may occur due to the local synthesis in the kidney, which includes a major fraction of NGAL in the urine and synthesis in distant organs and immune cells. Such conclusions, however, are not very convincing due to some uncertainty associated with the process of NGAL in the kidney at the level of glomerular filtration, tubular reabsorption and dilution of urine, and also the fact thatin situhybridization was performed in mice and that such methods are poorly reflect the ability of cells to production of the protein. Our results, however, do support the idea that tubular epithelial cells have the ability to produce NGAL, because the expression of mRNA and protein products induced under certain conditions related to the presence of the kidneys under stress conditions or under conditions of inflammation, such as observed during extracorporeal circulation. We found that the cytokine IL-1R will be the most powerful and effective incentive, which is compatible with the action of others, using epithelial cell lines of the lung. High levels of secretory proteins of neutrophils and cytokines, such as IL-β and TNF-α were observed during and after surgery on the heart in many previous studies. The results of the invention, therefore, demonstrated that NGAL present in the urine in many different who's forms and that are useful methods of analysis for the quantitative determination of NGAL in the urine, which take into account this diversity. Thus, in one of the embodiments the invention relates to the analysis, which preferably identifies NGAL originating from cells of the tubular epithelium, because the molecular structure of NGAL gives the impression that differ slightly from the structure of NGAL, originating from neutrophils. Such methods of analysis are, therefore, more specific and sensitive in the determination AKI and are of great benefit for patients with risk of impaired renal function.

Methods, devices and kits of the present invention is described based on specific embodiments, and in the section of examples illustrate specific aspects of the invention. However, it should be understood that additional embodiments of the aspects, variations and modifications of the invention may be carried out by the person skilled in the art without deviating the scope of the invention defined by the claims.

1. The method of determining in a sample of individual protein neutrophil origin of lipocalin associated with gelatinase (NGAL), which includes stages:
(a) determining the relative amounts of Monomeric, dimeric and heterodimeric forms associated with gelatinase neutrophil protein of lipocalin NGAL in the sample obtained from the organism of the individual, and
(b) comparison of the scientists in the determination of quantities where the overwhelming amount of Monomeric and/or heterodimeric forms of NGAL protein, compared with the dimeric form of the protein NGAL indicates that NGAL protein is derived from the kidney of an individual, whereas equal or predominant amount of the dimeric form of the protein NGAL compared to Monomeric or heterodimeric forms of NGAL protein, indicates that NGAL protein is derived from neutrophils of the individual.

2. The method according to claim 1 for definitions of acute kidney injury in the specified individual, where the overwhelming amount of Monomeric and/or heterodimeric forms of NGAL protein, compared with the dimeric form of the protein NGAL indicates that NGAL protein is derived from the kidney of an individual and that the individual has acute kidney injury, while equal or predominant amount of the dimeric form of the protein NGAL compared to Monomeric or heterodimeric form of NGAL protein, indicates that NGAL protein is derived from neutrophils of the individual and that the specified individual has no acute kidney injury.

3. The method according to claim 1 or 2, where the relative amount of NGAL protein in the sample is determined by bringing the sample into contact with the antibody to NGAL.

4. The method according to claim 3, where the antibody to NGAL includes polyclonal and/or monoclonal antibody to NGAL.

5. The method according to claim 4, where the antibody to NGAL includes two polyclonal antibody to NGAL.

6. The method according to claim 4, where the antibody to NGL includes two different monoclonal antibody to NGAL.

7. The method according to any of claims 4-6, where the stage of determining the relative amounts of Monomeric, dimeric and heterodimeric forms of NGAL protein comprises (i) providing a sample of body fluid of an individual in contact with the antibody to NGAL and detectable label to provide education complex NGAL protein in the sample with the antibody to NGAL and (ii) determining the relative amounts of the complexes formed between Monomeric, dimeric and heterodimeric forms of NGAL protein in the sample and the antibody, using a detectable label.

8. The method according to claim 3, where the stage of determining the relative amounts of Monomeric, dimeric and heterodimeric forms of NGAL protein comprises (i) providing a sample of body fluid of an individual in contact with the antibody to NGAL and detectable label to provide education complex NGAL protein in the sample with the antibody to NGAL and (ii) determining the relative amounts of the complexes formed between Monomeric, dimeric and heterodimeric forms of NGAL protein in the sample and the antibody, using a detectable label.

9. The method according to claim 7, where the amount of complex formed between NGAL protein in the sample and the antibody to NGAL, determine enzyme-linked immunosorbent assay (ELISA).

10. The method according to any one of claims 1, 2, 4-6, 8 and 9 for controlling the treatment of acute kidney injury, where the method includes anal is C the set of samples from the body of the individual, obtained before and after or during the course of treatment.

11. The method according to claim 3 for controlling the treatment of acute kidney injury, where the method includes the analysis of many samples from the body of the individual, obtained before and after or during the course of treatment.

12. The method according to claim 7 for controlling the treatment of acute kidney injury, where the method includes the analysis of many samples from the body of the individual, obtained before and after or during the course of treatment.

13. The method according to any one of claims 1, 2, 4-6, 8, 9, 11 and 12, where the sample is urine.

14. Kit for determination in the sample relative amounts of Monomeric, dimeric and heterodimeric forms associated with gelatinase neutrophil protein of lipocalin (NGAL), where the kit comprises a first antibody to NGAL, adapted for the formation of a complex with the protein NGAL in a sample of body fluid, the second antibody to NGAL protein, adapted for use in determining the amount of complex formed between the protein NGAL in a sample of body fluid and the first antibody to NGAL, and detektiruya tag adapted for use in determining the amount of complex formed between NGAL in a sample of body fluid and the first antibody to NGAL.

15. Set in 14, where the first and second antibody to NGAL include two different monoclonal antibody to NGAL; monoclonal anti-Christ. ate to NGAL and polyclonal antibody to NGAL; or two polyclonal antibody to NGAL.

16. Set on 14 or 15, where one of the above first or second antibody to NGAL adapt to the complex formation with the indicated Monomeric, dimeric or heterodimeric forms of NGAL and other designated first or second antibody to NGAL adapt to the complex formation only with the specified heterodimeric form of NGAL.

17. The set according to any one of p-16 in the method according to any one of claims 1 to 13.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: invention is a method of determining the nonspecific resistance of pathogenic microorganisms to antibiotics and the fact of the presence of bacterial biofilms on the basis of measurement of catalytic activity of phosphodiesterases cleaving the cyclic diguanosine monophosphate, with a threshold sensitivity of 50 pg/ml, comprising: 1) isolating the target-phosphodiesterase from lysed bacterial cells; 2) binding of phosphodiesterase with biotin-conjugated antibodies specific for non-catalytic domains of phosphodiesterase; 3) affinity purification of complexes formed by target-phosphodiesterase and biotin-conjugated antibody using paramagnetic particles containing neutravidin or its analogs that bind biotin; 4) interacting of the complexes of phosphodiesterase/biotin-conjugated antibody, immobilised on paramagnetic particles with complexes containing a-di-GMP in the form of G-quadruplex systems with intercalate dye, which is accompanied by decrease in the intensity while destruction of complexes of intercalate dye with c-di-GMP; 5) measurement of decrease of fluorescence upon hydrolysis with c-di-GMP and destruction of complex of c-di-GMP with intercalate dye, followed by quantitative estimation of the phosphodiesterase activity based on calibration curves made using known amounts of the recombinant enzyme of phosphodiesterase identical to the test target; 6) identification of increased level of phosphodiesterase activity detected by the test antibiotic-resistant bacterial strains capable of biofilm formation, as compared with the level of phosphodiesterase activity that can be detected for the control strains of bacteria of the same species not having the antibiotic resistance and the ability to form biofilms.

EFFECT: method enables to determine the nonspecific resistance of pathogenic microorganisms to antibiotics and to establish the fact of the presence of bacterial biofilms.

4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of immunology, namely to enzyme-immunoassay, in particular to a method of detecting forms of vascular endothelial growth factor (VEGF) with a size more than 110 amino acids in a biological sample. The method includes the following stages: contact and incubation of the biological sample with an uptake reagent, immobilised on a solid substrate, where the uptake reagent contains a monoclonal antibody, which recognises and specifically binds with residues, in quantity more than 110, from human VEGF; separation of the biological sample from the immobilised uptake reagents; contact of the immobilised molecular complex of the reagent of the uptake-target with detected antibody, which binds with VEGF domains, responsible for binding with KDR and/or FLT1 receptor, or which binds with an epitope in VEGF1-110; measurement of the level of VEGF110+, bound with reagents of the uptake, with application of means of detection for the detected antibody. Set of immune assay reagents for detection of VEGF110+ forms in the biological sample. An antibody 5C3, obtained from hybridoma 5C3.1.1 with a depositary number PTA-7737, with the said antibody 5C3 binding VEGF110+ forms, including VEGF121+. Hybridoma 5C3.1.1, deposited in ATCC with the depositary number PTA-7737, to obtain the monoclonal antibody 5C3.

EFFECT: application of the claimed invention makes it possible to increase accuracy of detecting VEGF isoforms, which must not include isoform VEGF110 and must obligatory include isoform VEGF121.

25 cl, 3 dwg, 2 tbl, 1 ex

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

FIELD: medicine.

SUBSTANCE: in the method for assessing action of biologically active substances on the antigen-antibody interaction based on sampling whole blood, stabilising it by an anticoagulant, adding a biologically active substance to the whole blood samples of the groups O(I)-AB(IV), incubating for 3-5 minutes, introducing standard monoclonal anti-A and anti-B antibodies, and 3-5 minutes later, rating the agglutination intensity relevant to the blood group.

EFFECT: improved assessment accuracy.

3 tbl

FIELD: medicine.

SUBSTANCE: flow cytofluorimetry is used to examine peripheral blood for the content of CB34+CB45+hemopoietic precursors on 1-3 pot-injury day, but not earlier than in 8 hours. If the content is lower than 3×106/l, an unfavourable clinical outcome is predicted, while the content 3×106/l and more shows a favourable clinical outcome of a moderate and severe craniocerebral injury.

EFFECT: using the method enables higher accuracy of the early prediction of clinical outcomes in the patients with the craniocerebral injury and the prescription of the adequate differentiated therapy.

3 ex

FIELD: medicine.

SUBSTANCE: what is described is a hybrid cultured cell strain of the animals Mus museums Sp2/0Ag14-SpBcG/APC-15/A3 that is a produced of a monoclonal antibody specific to human protein C (to hPROC). The strain is deposited in the Russian Collection of Vertebrata Cell Culture of the Institute of Cytology of the Russian Academy of Sciences, No. 733(D). What is described is a monoclonal antibody prepared of the strain, specific to hPROC and showing the conformational properties. It binds hPROC in the presence of calcium ions and does not bind it in the presence of chelating agents. What is presented is an immunosorbent on the basis of said antibody.

EFFECT: applicability of the strain for preparing the MCA to hPROC, and producing on its basis an immune-affine sorbent for hPROC purification and concentration.

3 cl, 2 dwg, 3 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: blood serum is examined for the content of acuity antibodies M and G to Herpesviridae family viruses, namely herpex simplex type 1 and 2 IgM; cytomegalovirus IgM; cytomegalovirus immediate-early protein IgG and IgM, Epstein-Barr virus core protein IgM, and Epstein-Barr virus early antigen IgG. If detecting the acuity antibodies simultaneously to two and more Herpesviridae family viruses, development of lung cancer is predicted.

EFFECT: method provides higher accuracy of prediction of development of the disease.

2 ex

FIELD: medicine.

SUBSTANCE: method of simultaneous immunochromatography analysis of PSA and CEA oncoantigens is offered. The analysis involves using a test strip which contains three segments A, B and C; the segment A overlaps 1-2 mm of the segment B. The segment A represents an inert porous carrier made of fibre glass ("ПЭД") with two reaction zones 1 and 2 applied on its surface. Mice monoclonal PSA and CEA antibodies conjugated with colloidal gold are respectively applied on zones 1 and 2. The conjugates are applied in the form of parallel strips in the centre of "ПЭД" perpendicularly to a fluid flow. The segment B represents nitrocellulose immobilised on a lavsan substrate with two test zones applied on its surface (monoclonal PSA and CEA antibodies in each), and a reference area (mice immunoglobulin antibodies).

EFFECT: test enables the simultaneous detection of the patients with the high blood serum PSA and CEA antigen contents in screening assays.

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.

SUBSTANCE: method for simultaneous determination of two analytes via bioluminescent molecular microanalysis involves treating the solid phase with a biospecific reagent, separating the unreacted liquid phase, treating the solid phase with an enzyme-containing conjugate, separating the liquid phase and the solid phase and analysing the solid phase, wherein the biospecific reagent for the first treatment of the solid phase is two immunoglobulins or one immunoglobulin with dual specificity or two oligonucleotides, and final treatment of the solid phase is carried out simultaneously with two enzyme-containing conjugates consisting of, respectively, recombinant Ca2+-dependent photoprotein obelins with different bioluminescence characteristics and molecules of different biospecificity, where the recombinant obelins used are obelin W92F; H22E and obelin Y138F, with subsequent analysis on a tablet bioluminometer with fast moving wideband colour filters with spectral and time separation of signals.

EFFECT: improved method.

3 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: ultrasound-assisted transcutaneous targeted needle liver-biopsy is used in the patients with acute destructive pancreatitis; that is followed by the immunohistological analysis of the biopsy micropreparations in parallel sections 5 mcm thick marked with monoclonal antibodies: CD 79a for identifying B-Lm and CD 68 for identifying MF. That is followed by optical microscopy at magnification x400 with morphometry of B-Lm periportal regions and MF lobe parenchyma; the results are further processed by automated image analysis systems. A relative area taken by the analysed cell elements in the micropreparation is determined by formula (A). If the value A for the B-Lm periportal regions falls within the range of 0.4 to 1.14, while the value A for the MF lobe parenchyma falls within the range of 0.89 to 2.95, a pre-infectious phase of acute destructive pancreatitis is stated. If the value A for the B-Lm periportal regions falls within the range of 1.41 to 4.98, while the value A for the MF lobe parenchyma falls within the range of 4.34 to 9.52, a phase of suppurative septic complications of acute destructive pancreatitis is stated.

EFFECT: using the method provides the well-timed correction of the therapeutic actions; the method is attended by a lower risk of the needle biopsy; it possess high accuracy and simplicity of implementation.

2 tbl, 2 ex, 6 dwg

FIELD: chemistry.

SUBSTANCE: detection system for detecting target molecules includes a sensor chip (1), having on its detecting surface (33) an immobilised target molecule or a capturing molecule for target molecules and a soluble reagent layer (5), having a labelled molecule for binding with the target. The group of inventions also relates to a sensor chip (1) and a method of detecting target molecules in a sample using said sensor chip.

EFFECT: high sensitivity and accuracy of analysis while cutting duration of analysis.

19 cl, 8 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: delimitation of a high-grade glioma invasion is ensured by imaging of an astroglial bank surrounding the high-grade glioma. An immunogenic recombinant human GFAP is prepared and used to immunise a Balb/C mouse; spleen B-lymphocyte of this mouse are recovered and fused with myeloma cells of Sp 2/0-Ag14 mice; hybridomas are produced. Supernatants of the prepared hybridomas are tested by immunochemical techniques for the presence of anti-GFAP antibodies used to select a hybrid cell clone producing the anti-GFAP antibodies able to distinguish GFAP in vivo. The anti-GFAP antibodies are cleaned from the supernatant of the selected clone and covalently bound with liposomal nanocontainers containing a diagnostic mark. The antibodies of the selected hybrid cell clone is modified by g-amino groups of lysine residues and incubated with the stelths-liposome solution. The prepared nanosystem is introduced in a patient's vascular bed, and the astroglial bank is imaged by the arrangement of the diagnostic mark in cerebral tissues.

EFFECT: method allows preventing relapses of high-grade gliomas after their surgical management by choosing an optimal extent of a pending surgery ensured by delimitation of a tumour invasion by means of imaging of the astroglial bank.

6 cl, 4 dwg, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biochemistry, more specifically to methods of carrying out immunoenzymometric and DNA-hybridisation analysis. A conjugate is proposed for bioluminescent molecular microanalysis, obtained from an enzyme, which is bonded to a biospecific reagent, where the enzyme used is Ca2+ regulated photoprotein obelin with an altered bioluminescent spectrum: recombinant obelin W92F; H22E or recombinant obelin Y138F. The method for simultaneous detection of two analytes through bioluminescent molecular microanalysis involves treating a solid phase with biospecific reagents, separation of unreacted liquid phase, treatment of the solid phase with an enzyme containing conjugate, separation of the liquid and solid phases and analysis of the solid phase. The biospecific reagent used for the first treatment of the solid phase is two immumoglobulins or one immunoglobulin with double specificity or two different oligonucleotides. Final treatment of the solid phase is done simultaneously with two conjugates, which respectively consist of recombinant obelins with different bioluminescent spectra and molecules with different biospecificity, where the recombinant obelins used are W92F;H22E and recombinant obelin Y138F with subsequent analysis on a double-channel bioluminometre with broad-band light filters.

EFFECT: use of this invention allows for accurate, fast, universal and easy realisation of the method of bioluminescent molecular microanalysis.

4 ex, 3 dwg, 2 cl

FIELD: biotechnologies.

SUBSTANCE: claimed is test-system for determining specific activity of anti-rabies serums and immunoglobulin. Test-system contains nitrocellulose membrane with applied on its surface positive (branch standard sample of anti-rabies immunoglobulin specific activity) and negative (normal horse serum) controls, de-ionised water for washings, sample dilution, conjugate and development (visualisation) system. As conjugate, used is gold hydrosol with particle size 15-17 nm, sorptially connected with inactivated fixed rabies virus of production strain "Moskva-3253" from rabbit virus-containing brain suspension or rabies virus glycoproteide.

EFFECT: extending possibility of determining anti-rabies serum and immunoglobulin specific activity in production of heterological anti-rabies immunoglobulin.

6 cl, 2 tbl, 2 ex

The invention relates to medicine, namely to Allergology, and can be used to identify sensitization to allergens in adults and children
The invention relates to the field of veterinary medicine, namely to laboratory diagnosis
The invention relates to the field of veterinary medicine, namely to laboratory diagnosis

FIELD: measurement equipment.

SUBSTANCE: invention relates to a device and method for quantitative measurement of analyte using a chamber. Namely, for acquisition of identification code data required to obtain accurate analyte analysis result. The method involves acquisition of data as a result of interaction of analyte using a chamber, without any additional equipment; reading of the identification code and result of analyte interaction. The measuring device includes the following: a chamber for catching a detection zone by a chamber of analytical set, which includes analyte interaction result obtained by interaction of analyte, and identification code of analytical set; a unit for processing of images to detect an image of analyte interaction result and an image of identification code from images of the detection zone by the analytical set chamber caught by the chamber; a read-out unit for reading of an image of analyte interaction result and an image of identification code; a control unit to provide the possibility of processing the read-out result of the image of the interaction result of analyte subject to processing; and an output unit for output of a final result on analyte, which is obtained by the control unit.

EFFECT: invention ensures an accurate result generation.

18 cl, 9 dwg

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