RU133313U1 - DIAGNOSTIC TEST SYSTEM IN THE FORMAT OF AN IMMUNOCHIP AND METHOD FOR SEROLOGICAL DIAGNOSTICS OF IKSODA TICK BORRELIOSIS - Google Patents

Abstract

1. A diagnostic test system in the format of an immunochip for serological diagnosis of tick-borne ixodic borreliosis, including an immunosorbent in the form of a slide, divided into Erreys, each of which is designed to analyze one sample, or as an immunological tablet, each well of which is designed to analyze one sample, with slots containing diagnostically significant antigens / synthetic peptides belonging to the Borrelia burgdorferi sensu lato complex immobilized within the boundaries of each Herrea or well of the plate of the tablet, and e auxiliary control spots for assay standardization immunochip and checking the validity of the test system, a conjugate consisting of a mixture of secondary antibodies to human IgG and anti-human IgM, modified in two different spectral characteristics fluoroforovymi krasitelyami.2. A diagnostic test system in the format of an immunochip for serological diagnosis of tick-borne ixodic borreliosis according to claim 1, additionally containing reagents necessary for conducting and validating the analysis. 3. The diagnostic test system in the immunochip format for serological diagnosis of tick-borne ixodic tick-borne borreliosis according to claim 1, where each slide erray or well of the tablet contains a combination of separately immobilized slots, each of which contains at least one diagnostically significant recombinant antigen / synthetic peptide selected from the group : Borrelia garinii p 100, Borrelia afzelii p100, Borrelia garinii p41, Borrelia afzelii p41, Borrelia garinii BBK32, Borrelia afzelii BBK32, Borrelia garinii p17, Borrelia afzelii p17, Borrelia garinii OspC, Borrelia afzelii Ospelii, Borrelia afzelii Ospelii Borrelia afzelii VlsE, Borrelia garinii VlsE. 4. Diagnostic test system for

Description

The invention relates to the field of clinical laboratory diagnostics, biotechnology and medicine, in particular, to a diagnostic test system in the format of an immunochip (biological chip) and a method for serological diagnosis of tick-borne tick-borne borreliosis.

State of the art

Lyme borreliosis (LB), Lyme disease, or tick-borne ixodic borreliosis (IKB) is a transmissible polysystemic disease that leads to damage to the musculoskeletal system, nervous and cardiovascular systems, and skin, which occupies one of the leading places in terms of breadth of distribution and incidence rate. among natural focal diseases. Natural foci of Lyme borreliosis are widespread in North America, Europe and Asia, and are confined mainly to forest landscapes of the temperate climatic zone. The disease is recorded on a large territory of the Russian Federation. Due to the relatively high incidence rate (compared with other natural focal vector-borne infections) and often severe course, LB is one of the urgent problems of modern infectious diseases in Russia.

The causative agent of tick-borne borreliosis is the Borrelia burgdorferi sensu lato complex, and the carriers are ixodid ticks. Currently, more than 10 genomic groups belonging to the complex Borrelia burgdorferi sensu lato, which are unevenly distributed throughout the globe, have been isolated. The pathogenic role of three genre types of Borrelia from the group of B.burgdorferi sensu lato (B.burgdorferi sensustricto, B.garinii, B.afzelii) in the occurrence of a disease in a person has been proved. Genovids B.lusitaniae, B.valaisiana, B.miyamotoi have good reasons to supplement this list. It should be noted that all these genotypes are distributed in Russia. The specific weight of each genovid in the epidemiology of tick-borne borreliosis in different geographical regions is different. Simultaneous infection with different genes of borrelia is possible, which leads to a peculiar clinical picture of the disease, different from monoinfection (borreliosis mixed infection). The observed differences in the clinical picture of Lyme disease in patients at various points of the nosoareal of this infection may be based on the genetic heterogeneity of the B. burgdorferi sensu lato complex.

Diagnosis of LB is usually carried out on the basis of an epidemiological history (visiting the forest, tick sucking), as well as the clinical manifestations of the disease, the main of which is annular erythema migrans. However, up to 45% of cases of LB occur in an erythema-free form, and in some patients, migratory erythema goes unnoticed. Clinical diagnosis of non-erythema forms of LB presents significant difficulties and is possible, as a rule, only at later stages, when the disease begins to manifest itself in the form of any organ lesion syndrome.

The presence of infection is detected using laboratory methods, which can be divided into 2 groups:

1. direct methods for the detection of borrelia, their antigens or nucleic acids in biological samples of the person being examined (blood, cerebrospinal fluid, synovial fluid, biopsy samples), for example, by PCR;

2. serological methods - determination of specific antibodies to B. burgdorferi antigens in biological fluids of patients.

The PCR method has been successfully used to determine tick borrelia infection, however, analysis with its help of various biological samples of patients with LB does not give reliable results. Currently, PCR is not recommended as an independent screening test, this method is also unsuitable for evaluating the effectiveness of antibiotic therapy. Sometimes PCR is used for additional examination of a patient with clinical signs of LB, but with a negative or doubtful result of serological tests. However, even in these cases, a negative result of the determination of B. burgdorferi nucleic acids in biopsy specimens, synovial or cerebrospinal fluid does not prove the absence of a borreliosis infection in the examined patient.

The most common methods of serodiagnostics for ICD are enzyme-linked immunosorbent assay (ELISA), immune blotting, immunofluorescence method (RNIF, indirect immunofluorescence reaction). In 1994-1995, in the United States and European countries, to improve the reliability of serodiagnosis of borreliosis, it was recommended to use a two-stage test scheme for testing blood serum / cerebrospinal and / or synovial fluid. At the first stage, using RNIF (and more often, ELISA), screening studies of the sera of patients with suspected borreliosis are carried out, at the second stage, samples that give positive or doubtful results during screening are re-tested by the immunoblot method, which surpasses these methods in specificity. According to the results of the immunoblot, by analyzing the spectrum of specific antibodies to specific Borrelia proteins, it is possible to indirectly judge the stage of development of the disease (early or late manifestations of LB), as well as the recurrence of the disease or its relapse within the same epidemic season.

In 60-90% of patients with lesions of the central nervous system with LB, specific antibodies can also be determined in the cerebrospinal fluid, however, their detection is not a decisive factor for confirming borreliosis of the central nervous system, since antibodies can penetrate into the subarachnoid space from the blood. An immunoblot for these purposes can be used in cases where there is an equalization of the concentrations of specific antibodies in the cerebrospinal fluid and blood serum. The determination of a wide range of antibodies in cerebrospinal fluid, specific for different proteins of borrelia, in the absence of some of them in the study of blood serum indicates a predominantly intrathecal synthesis of antibodies.

In an immunoblot, nitrocellulose or nylon membranes are used for analysis onto which B. burgdorferi cell lysate proteins separated by polyacrylamide gel electrophoresis are transferred. Moreover, depending on the conjugate used, it is possible to determine IgM, IgG or total antibodies to a large number of borrelia proteins in the tested sera at the same time. Using the blot, it is possible to identify specific antibodies to specific borrelia proteins, to study the dynamics of the production of antibodies to various diagnostically significant proteins, which allows clinicians to predict the course of the disease. A human humoral immune response to borreliosis infection can be represented by antibodies to more than twenty B. burgdorferi antigens.

Given that the composition of antigen proteins and their ratio can vary significantly not only among borrelia of different genovids (in which the heterogeneity of a number of surface proteins reaches 40%), and the level of expression of certain immunodominant lipoproteins and the spectrum of antibodies significantly change during the development of infection in B. burgdorferi in infected patients, it can vary depending on the genus of the pathogen, as well as in one patient at different stages of the disease. So, often at stage I of LB, class M immunoglobulins against flagellin, OspC appear in the patient’s body. The simultaneous detection of a wide spectrum of antibodies to antigens (p100, p75, Oms66 / p66, OspA, BmpA / p39, p18, p21) allows us to draw a conclusion about a fairly long period of time that has passed since the infection (even in cases of asymptomatic disease course). It should be noted that the detection of antibodies to B. burgdorferi in the blood of the examined patients is not necessarily accompanied by a clinical manifestation of LB, and vice versa - in 30% of patients with borreliosis with annular erythema migrans, specific IgM in the first stage of LB can not be determined. With neuroborreliosis, in 80% of cases, antibodies in the cerebrospinal fluid of patients appear 2 weeks after a tick bite, and in 100% of cases after 7 weeks. In this case, the test for the presence of antibodies in the patient's blood may be negative. As the disease develops, the spectrum of human antibodies to the pathogen antigens broadens, stage II LB IgG (progressive, disseminated) is produced both to flagellin, OspC and VlsE, as well as to new ones: p39, p58, p60, p66, p75. It should be noted that in some patients, IgM to OspC protein can be detected in the blood for several months. At the III stage of borreliosis (chronization), the immune response is characterized by an even wider spectrum of specificity of the antibodies produced. During this period, more than 80% of the synthesized class G immunoglobulins react with the proteins p83 / 100, p58, p43, p39, p30, p21, Osp17 and p14. However, antibodies to the OspC protein are very rare, since the expression of this lipoprotein during chronic disease is significantly reduced. It was found that if at an early stage of LB patients who have no antibodies to B. burgdorferi (or are detected in a low concentration) received an effective course of antibiotic treatment, then their antibody level does not subsequently increase. However, if antibiotic therapy was performed for patients with LB with positive results of serological tests, then the concentration of specific antibodies after treatment may remain at a detectable level for a long time (from several months to several years). It was shown that antibodies to B. burgdorferi in patients with neuroborreliosis continue to be determined in the cerebrospinal fluid for a year after successful treatment. In cases where after the treatment and discharge of the patient from the hospital, he again shows signs of LB, against the background of an increased content of IgM and IgG (to flagellin), then this should be regarded as cases of reinfection.

The use of diagnostic test systems, which are based on a limited set of antigens, can lead to a situation where a negative result of the study is due to the fact that the human immune system simply does not produce specific antibodies against these antigens, since borrelia that persist in the human body do not have these proteins. The efforts of many research laboratories and companies specializing in the production of diagnostic tools are aimed at improving methods for diagnosing LB. To this end, intensive research is underway to select the optimal composition of B. Burgdorferi proteins used for ELISA, to search for new immunologically significant borrelia antigens, and to study their main antigenic determinants. In the immune blot, as a rule, the spectrum of antigen is represented most widely in comparison with enzyme immunoassay systems. Moreover, each antigen is immobilized individually, and not in a mixture, which often increases the sensitivity and specificity of the test as a whole, because minimize the effect of desorption, partial overlap of important protein epitopes.

Due to the characteristics of the immune response in LB, the effectiveness of detecting antibodies to B. burgdorferi in patients even with a combination of modern serological tests varies significantly depending on the stage of the disease. So, at an early stage of the disease, it is possible to identify up to 50% of patients with borreliosis, at stage II - 70-90%, and only at stage III - up to 100%. Since a negative serological test result in the early stages of the disease does not prove that there is no infection, it is recommended that patients with suspected LB be tested for antibodies to B. burgdorferi after 2-4 weeks with a fresh sample. If specific antibodies are detected, the result of serodiagnosis is evaluated as positive, confirming borreliosis infection.

Differential definition of antibodies by classes is important in the diagnosis of borreliosis. As a rule, IgG antibodies appear in the period from several weeks to months after infection. In the early stages of infection, they are often not detected. In this case, the repeated sample should be checked some time later. Patients in the 2nd or 3rd stage of the disease usually give a positive IgG antibody test result. IgM antibodies usually appear 2–3 weeks after the onset of the disease. Antibody titer is often reduced within a few weeks to months after recovery. It is also possible that the antibody titer remains constant for several years. An important condition for serological detection of antibodies is the normal immune status and ability of the immune system. Some cases of seronegative Lyme borreliosis in patients with a progressive course of the disease and certain clinical manifestations of borreliosis are largely due to the immunosuppression state of these patients.

Thus, of all the currently available serological diagnostic tests, only the immunoblot has the highest information content, because allows you to determine the spectrum of antibodies to significant antigens. For example, US 2006281139 A1 (VIRAMEDBIOTECHAG) 12/14/2006 discloses kits for the diagnosis of borreliosis by immune blotting.

However, the use of an immunoblot far from completely solves the problems of serodiagnosis of LB, since:

- the results are often evaluated visually, so it is subjective and depends on the qualifications of the laboratory staff;

- the criteria for assessing the results of immunoblot are complex, they differ in the USA, where only one genus of B. burgdorferi sensu stricto is circulating, and in Europe, where the genes of B. afzelii and B. garinii are more heterogeneous in antigens, and manufacturers use various Borrelia strains;

- it is not possible on the same strip to simultaneously and separately determine the spectrum of antibodies by classes,

- the use of immunoblot significantly increases the cost of serological analysis.

Closest to the present invention is the patent application "Borrelia burgdorferi cell envelope protein array" (WO 2009/131665 A1 (RESEARCH FOUNDATION OF STATE UNIVERSITY OF NEW YORK) 2009.10b29) (prototype). This document provides a method for determining the presence of antibodies to Borrelia burgdorferi proteins, as well as a method for diagnosing Lyme disease. The proposed method is carried out using chips.

However, the proposed protein chip does not allow specific antibodies of different classes G (IgG) and M (IgM) to be simultaneously detected on the same immunosorbent in the same erray and spot, which is achieved in the present invention using a conjugate consisting of a mixture of secondary antibodies to human IgG and antibodies to human IgM, modified by two different spectral characteristics of fluorophore dyes.

Although the example of WO 2009/131665 on page 8 indicates the use of fluorophore-conjugated antibodies, namely, goat antibodies to human IgG / IgM / IgA conjugated to Csu5 and goat antibodies to mouse IgG immunoglobulins, conjugated to Cy3, however, such the conjugate with Cy5 allows you to determine the total IgG / IgM / IgA antibodies and is not intended for the differential diagnosis of IgG and IgM, as proposed in the present invention. And the second conjugate with Cy3 is used to calculate the ratio of Cy5 intensity / Cy3 intensity (protein / His-Tag) for each protein, which is used to assess the intensity of each of the studied antigens.

Thus, the technical task of the present invention is to expand the arsenal of new generation diagnostic tools intended for differential detection of the spectrum of antibodies G and M to the spectrum of antigens of the causative agent of borreliosis for a more accurate, more informative and specific diagnosis of ICD at different stages of the disease. The technical result consists in the creation of a diagnostic test system in the format of an immunochip and in the implementation of the indicated purpose of the test system for the simultaneous and differential detection of the spectrum of antibodies to the diagnostically significant immunogenic proteins of Borrelia classes G (IgG) and M (IgM), which is confirmed by the materials of this application.

Disclosure of invention

In the framework of the present invention, the following terms are used:

biochip - a diagnostic test system that includes an immunosorbent, which is a small matrix (slide, bottom of the plate well, strip) with a set of individual microdots or slots (from the English spot - spot) with a diameter from 10 to 500 microns of biologically active samples substances (oligonucleotides, antigens, antibodies, enzymes, allergens, etc.) and all the necessary reagents for complex serological analysis (immunomicrochip) or hybridization analysis (DNA chip).

immobilization is the process of irreversible “sewing” of biologically active substances (antigens, antibodies, allergens, etc.) onto the surface of the solid phase (immunological plate, glass slide, membrane strip, etc.).

immunosorbent (in chip format) - a sorbent (slide with an activated chemical groups or their derivatives surface, immunological plate, strips) with functional biological molecules immobilized as a result of sorption on it (antigens, antibodies, oligonucleotides, etc.)

errey (from the English array) - a limited area on the immunosorbent in the form of a slide, intended for the placement of one analyzed sample, is an analogue of the immunological plate well for ELISA or strip in immune blotting. Inside each herrey there is a necessary set of antigens, internal controls, markers for printing.

spot - a place of immobilization of antigens / antibodies / oligonucleotide or any other biological material on errey.

All of these terms, in principle, are known to the person skilled in the art and are given here with an illustrative purpose for a better understanding of the essence of the proposed invention.

In accordance with the present invention, a diagnostic test system in the format of an immunochip and a method for serological differential diagnosis of tick-borne tick-borne borreliosis are provided.

The diagnostic test system in the immunochip format for serological differential diagnosis of borreliosis consists of an immunosorbent with antigens and / or synthetic peptides immobilized separately on it, belonging to the Borrelia burgdorferi sensu lato complex, conjugate and reagents necessary to detect the antigen-antibody complex.

On the immunosorbent, auxiliary controls are additionally immobilized. Auxiliary controls are antibodies to human IgG immunoglobulin and / or antibodies to human IgM immunoglobulin and / or human IgG immunoglobulin and / or human IgM immunoglobulin and / or internal negative control and / or markers of Errei boundaries.

An immunosorbent is a slide in the form of a glass or plastic base with a modified surface or coated with nitrocellulose.

The immunosorbent, made in the form of a slide, is divided into Errei, each of which is designed to analyze one sample.

An immunosorbent can be an immunological plate, each well of which is designed to analyze one sample.

As reagents necessary for conducting and validating the analysis, the diagnostic test system may contain reagents for detecting the antigen-antibody complex, for example, such as solutions for diluting control and test samples, a solution for diluting the conjugate, a solution for washing the immunosorbent, a positive control sample (K +), negative control sample (K-).

The method of serological differential diagnosis of borreliosis is carried out using a diagnostic test system in the format of an immunochip, according to which a solution is added to the immunosorbent with antigens separately immobilized on it, control and test samples are diluted, control and test samples are introduced, and then the resulting mixture is incubated at a temperature 20-42 ° C for 15-60 minutes to form an antigen-antibody complex, the immunosorbent is washed, then a conjugate solution is added to the immunosorbent ata from a mixture of antibodies to human IgG and antibodies to human IgM modified with various spectral characteristics of fluorophores, and incubation is carried out at a temperature of 20-42 ° C for 15-60 minutes, the immunosorbent is washed, dried, and then the resulting antigen complex is detected. antibody.

The detection of antibodies to specific antigens is carried out on the basis of assessing the intensity of fluorescence signals at the sites of localization of an antigen on an immunosorbent.

Differential detection of antibodies of various classes is carried out through the use of a conjugate consisting of a mixture of antibodies to human IgG and antibodies to human IgM modified with fluorophores corresponding to different spectral characteristics, and the results are recorded using a multichannel fluorescence scanner when the corresponding channels are activated.

The diagnostic test system contains an immunosorbent with separately diagnostically significant antigens and / or peptides immobilized on it, belonging to the Borrelia burgdorferi sensu lato complex, the conjugate and reagents necessary to detect the antigen-antibody complex. On the immunosorbent, internal controls can be additionally immobilized.

As reagents for detecting the antigen-antibody complex can be used:

- a solution for diluting the test and control samples (for example, 1% solution of casein and 2% bovine serum albumin in phosphate-buffered saline),

- a solution for diluting the conjugate (for example, a 1% casein solution in phosphate-buffered saline with 0.1% tween-20),

- solution for washing the immunosorbent (for example, phosphate-buffered saline with a content of 0.1% tween-20),

- positive control sample (K +) - inactivated by heating a pool of sera or plasma from patients with IKB, containing antibodies of classes G and M,

- negative control sample (K-) - a pool of serums or blood plasma inactivated by heating from practically healthy donors, not containing antibodies of classes G and M to the causative agent of ICD.

A preferred embodiment of the invention is the simultaneous analysis of a group of test samples using an immunosorbent, which contains from 12 to 16 erreys (identical zones) with immobilized significant antigens of borreliosis pathogens, and auxiliary controls. Each herray serves to set up one test or control sample. When conducting an analysis on one herray, a positive control sample of the test system (K +) is introduced to study the validity of the test system. Another Herrey is occupied by a negative control sample of the test system (K-). The remaining unoccupied Herrey contribute test samples.

As the solid phase for immunochips, microscopic slides 25 ± 1 mm wide, 75 ± 1 mm long and 1 mm thick with a modified surface (may contain epoxy, aldehyde, amino groups, polymers, etc.) or coated with nitrocellulose can serve. To exclude the confluence of samples, erray is separated by special devices - sealant frames. There are commercially available frames and slide holders, for example, Whatman, USA, which share an herray in the analysis.

Printing technology allows you to apply biological substances also to the bottom of the well of an EIA polystyrene tablet, which makes it possible to use the tablet as an immunosorbent, moreover, one well of the tablet corresponds to one errey and is intended for analysis of one sample.

With the help of modern printing systems - plotters - diagnostically significant borrelia antigens, auxiliary controls are immobilized (adsorbed) on the surface of the solid phase in the form of individual dots or slots ranging in size from 50 to 500 microns. To eliminate errors during printing (skipping printing) or during the analysis on an immunochip each antigen, auxiliary controls are immobilized separately in at least two repetitions. When analyzing the results of testing the samples, the average slots value of the fluorescence signal is taken into account. Auxiliary controls are:

- internal negative control (sorption buffer) is used to control print quality;

- human IgG immunoglobulin and / or human IgM immunoglobulin are used to control the introduction of a conjugate solution into errey;

- antibodies to human IgG immunoglobulin and / or antibodies to human IgM immunoglobulin are used to control the introduction of the sample into errey;

- markers of the boundaries of the Herrey, which are immobilized along the boundaries of each Herrey, allow after the analysis to visually observe the location of the Herrey and to control the imposition of the "grid" when the operator uses the program for calculating the results.

As markers of the boundaries of errei most often immobilize any non-specific protein for the diagnosis of borreliosis, for example, BSA, modified by one of the fluorophores.

Brief Description of the Figures

The invention is illustrated by the following figures of graphic images:

The Figure 1 shows an embodiment of an immunosorbent diagnostic test system for serological differential diagnosis of tick-borne ixodic borreliosis in the form of a slide containing 12 herrei.

Figure 2 shows an enlarged herray.

Immunosorbent is a slide 1 (Figure 1) in the form of a glass or plastic base with a modified surface, which is divided into Herrey 2, located at the same distance from each other. The boundaries of 3 Herrey 2 to prevent the fusion of the samples provide the imposition of a special frame (the frame in the drawing is not shown). Herrey 2 (Figure 2) contain spots 4 in the form of individual points.

The figure 3 shows an example of a fluorescence image of the profiles of errey on different channels for a patient with a positive serological diagnosis of tick-borne tick-borne borreliosis (stage 3), the blood serum of which contains antibodies of class G to ICD and does not contain antibodies of class M

The implementation of the invention

Diagnostically significant antigens / synthetic peptides belonging to the complex Borrelia burgdorferi sensu lato, the causative agent of borreliosis, are separately immobilized on the immunosorbent. In particular, known antigens can be attributed to them: Borrelia garinii p 100, Borrelia afzelii p 100, Borrelia garinii p41, Borrelia afzelii p41, Borrelia garinii BBK32, Borrelia afzelii BBK32, Borrelia garinii p17, Borrelia afzelii p 17, Borrelia gari Borrelia afzelii OspC, Borrelia afzelii p39, Borrelia afzelii p58, Borrelia afzelii VlsE, Borrelia garinii VlsE. The above list of antigens is not limiting, and other diagnostically significant antigens / synthetic peptides are known to a person skilled in the art.

For example, infection of ticks Borrelia afzelii and Borrelia garinii is most often found in the territory of the Russian Federation, and in this case it is advisable to include significant antigens of these genovids in the composition of the immunosorbent. In order to minimize the effect of overlapping immunogenic epitopes of antigens and increase the sensitivity of the immunochip, it is most justified to place the above antigenic groups individually. According to our observations, the immunoreactivity of proteins of different geno types in case of monoinfection (the patient is infected with only one geno type) is different and, as a rule, the level of antibodies to this geno type prevails.

The following is the clinical / diagnostic significance of the main borrelia antigens according to specialists in the study of immunopathogenesis of IKB.

p100 is a protein of membrane vesicles, immunogenous, with a chronic stage, IgG is predominantly found in it, highly specific. It is a typical marker of the late or chronic phase of the disease. In some cases, in the acute stage of Lyme disease, IgM antibodies of a given antigen are determined. In this situation, we can assume a high probability of Lyme disease becoming chronic.

VlsE-exposed surface lipoprotein (Variable major protein-like sequence), the highly specific domain of IR6 is often used in ELISA.

p58 - membrane protein, immunogen.

The p41int antigen is immunogenic, an internal specific site of flagellin. In the early stage of Lyme disease, in many cases only IgM antibodies are observed against the p41 antigen and the highly specific OspC antigen, which makes it possible to diagnose the early stage of Lyme disease.

p39 = BMp - the main protein of the membrane encoded on the chromosome, weakly immunogenic, but highly specific, often used for the production of ELISA tests in Europe.

BbK32 is a fibronectin-binding protein, localized in the outer membrane, immunogenous, specific, determined at all stages of the disease.

OspC-lipoprotein of the outer membrane of Borrelia, an early phase marker.

p17 = DbpA (Decorin-binding protein A) is a sensitive and specific antigen. Additionally, auxiliary controls are immobilized within each herray.

In order to determine antibodies of various classes on a single immunosorbent in accordance with the present invention, it is proposed to use a conjugate that is a mixture of anti-species antibodies to human IgG and to human IgM, which are modified correspondingly with different spectral characteristics of fluorophores. Antibodies to human IgG are modified, for example, with a Cy5 fluorophore, and antibodies to human IgM are modified, for example, with a Cy3 fluorophore. During scanning, when the corresponding channels of the fluorescence scanner are activated, differentiation of antibodies of different classes becomes possible. To do this, the scanner program indicates the type of fluorophores used in the conjugate or the corresponding parameters of the excitation and emission wavelengths, and the scanner automatically activates these channels.

Any fluorophores active in the visible light range can be used as fluorescent dyes, for example, SybrGreenI, FAM, TET, JOE, VIC, HEX, ROX, TAMRA, Cy3, Cy3.5, Cy5, Cy5.5, OregonGreen ^TM , CALRed ^TM , Red 640, Texas Red.

A method for serological diagnosis of borreliosis using a diagnostic test system in the form of an immunochip is as follows.

An immunosorbent with previously immobilized separately significant antigens belonging to the Borrelia burgdorferi sensu lato complex is added with a solution for dilution of control and test samples, after which control and test samples are added (preferred final dilution of the introduced samples to a dilution solution of -1: 10). After this, the resulting mixture is incubated at a temperature of 20-42 ° C for 15-60 minutes (preferably using a thermal shaker with a platform rotation of 500 rpm) to form an antigen-antibody complex. Further, the immunosorbent, in order to remove unreacted components, is washed at least one to two times with a solution for washing the immunosorbent, for example, with 1-time phosphate-saline buffer (pH 7.2) with the addition of 0.1% Tween-20.

A conjugate solution from a mixture of anti-IgG and human anti-IgM modified with various spectral characteristics of fluorophores, previously prepared from the conjugate and the conjugate dilution solution, is introduced onto the immunosorbent. Incubation is carried out at a temperature of 20-42 ° C for 15-60 minutes. To ensure the best result, incubation is carried out using a thermal shaker with a platform rotation of 500 rpm. After that, the immunosorbent is washed with a working solution for washing the immunosorbent and dried for subsequent detection of the analysis results.

Additionally, at least one antigen and / or peptide selected, for example, from the group of Borrelia garinii p 100, Borrelia afzelii p 100, Borrelia garinii p41, Borrelia afzelii p41, Borrelia garinii BBK32, Borrelia afzelii BBK32, can be separately immobilized on an immunosorbent. , Borrelia garinii p17, Borrelia afzelii p17, Borrelia garinii OspC, Borrelia afzelii OspC, Borrelia afzelii p39, Borrelia afzelii p58, Borrelia afzelii VlsE, Borrelia garinii VlsE, as well as auxiliary controls (antibodies to immunoglobulin or IgG / IgG human and / or human IgG immunoglobulin and / or human IgM immunoglobulin and / or negative internal control and / or and markers Erra boundaries).

Detection of the resulting antigen-antibody complex is carried out using a multi-channel fluorescence scanner for biochips when the corresponding channels are activated. Processing of the scan results is carried out in accordance with the instructions for the software used that came with the scanner. The detection of antibodies is carried out on the basis of assessing the intensity of fluorescence signals in the localization of specific antigens / peptides on the immunosorbent.

Next, calculate the value of the critical level of the coefficient of fluorescence (cut off) for each of the immobilized antigens. The value of the critical level of the fluorescence coefficient for each antigen is calculated, for example, by multiplying the fluorescence coefficient in errey with a negative control sample (K-) added by the alpha value, which is set by the biochip manufacturer for each series of chips by statistical analysis. Positive factors are calculated for each test sample for all immobilized antigens. The coefficient of positivity is the ratio of the fluorescence coefficients to the critical level of the fluorescence coefficient (cut off). Based on the value of the coefficients of positivity, conclusions are drawn about the presence or absence of antibodies to immobilized antigens.

The results of the analysis on the chip are considered valid (reliable) if the following conditions are met:

- on one specific channel, for example, Cy5 (determination of class G antibodies), spots corresponding to immobilized human IgG immunoglobulin should fluoresce within each erreia. The fluorescence signal of these spots should be at least three times higher than the background fluorescence level on errea,

- on the second specific channel, for example, Cy3 (determination of class M antibodies), spots corresponding to immobilized human IgM immunoglobulin should fluoresce within each erreia. The fluorescence signal of these spots should be at least three times higher than the background fluorescence level on errea,

- the level of spot fluorescence corresponding to the localization of the internal negative control (sorption buffer) should not exceed the level of fluorescence of the background of erray more than three times,

- the fluorescence level of all spots corresponding to the localization of antigens on specific channels of Su5 and Su3 in erray with a negative control sample (K-) of the test system introduced should not exceed the level of background fluorescence on erray more than three times.

Next, differential diagnosis of antibodies to ICD is carried out in accordance with the instructions for use with the test system. The criteria for interpretation in the immunochip for the presence / absence of antibodies to immunoglobulins of class G (IgG) and class M (IgM) see tables 1 and 2.

Table 1 Interference criteria in the immunochip for the presence / absence of antibodies to class G immunoglobulins (IgG) on the Cy5 channel. negative result 1. for all antigens of Borrelia afzelii and Borrelia garinii revealed values of positive coefficients of less than 1.1. 2. for p17 antigens of Borrelia afzelii and / or Borrelia garinii, positivity coefficients greater than or equal to 1.1 were detected positive result The presence of one of the following specific antibody spectra:

1. for all Borrelia afzelii and Borrelia garinii antigens, positivity coefficients greater than or equal to 1.1 were detected; 2. for VlsE antigens Borrelia afzelii and / or Borrelia garinii, values of positivity coefficients greater than 1.1 were detected (with or without antibodies to other significant borrelia antigens with positivity coefficients greater than or equal to 1.1); 3. positivity coefficients greater than or equal to 1.1 for at least two Borrelia afzelii and / or Borrelia garinii antigens from the group: p100, p41, p17, OspC, p58, Bbk32, p39 were revealed. uncertain result positive coefficients were found to be greater than or equal to 1.1 for one Borrelia afzelii and / or Borrelia garinii antigen from the group: p100, p41, OspC, p58, Bbk32, p39. Note: If there is an uncertain result or for the purpose of dynamic monitoring of the development of the disease, the results of the treatment of Lyme disease, it is recommended to re-test in the immunochip a new sample of human serum / plasma after 1-3 months at the same time as placing the sample taken earlier and stored under freezing conditions (thawing is allowed - freezing no more than two times).

Table 2. Interference criteria in the immunochip for the presence / absence of antibodies to class M immunoglobulins (IgM) on the Cy3 channel. negative result for all Borrelia afzelii and Borrelia garinii antigens, positivity ratios of less than 1.1 were detected. positive result The presence of one of the following specific antibody spectra: 1. for all Borrelia afzelii and Borrelia garinii antigens, positivity coefficients greater than or equal to 1.1 were detected; 2. For OspC antigens Borrelia afzelii and / or Borrelia garinii, values of positivity coefficients greater than 1.1 were detected (with or without antibodies to other significant borrelia antigens with positivity coefficients greater than or equal to 1.1); 3. values of positivity coefficients greater than or equal to 1.1 were revealed for at least two Borrelia afzelii and / or Borrelia garinii antigens from the group: p41, p17, VlsE. uncertain result positive coefficients were found to be greater than or equal to 1.1 for only one Borrelia afzelii and / or Borrelia garinii antigen from the group: p100, p41, p17, p58, Bbk32, p39. Note If an acute stage of infection is suspected, repeated testing in the immunochip of a human serum / plasma sample taken after 10-14 days is recommended

after the initial screening. In acute borreliosis, class M antibodies, as a rule, persist for up to 3 months (however, in some patients, class M antibodies persist for years). It is recommended that a new sample of human serum / plasma be tested in the immunochip with the simultaneous placement of a sample taken earlier and stored under freezing conditions (thawing-freezing no more than two times is allowed).

For a better understanding of the invention, the following are examples of its implementation.

Example 1

Differential serological diagnosis of ixodic tick-borne borreliosis on the example of a blood serum of a patient A.A.

Patient A.A.I. - IKB, stage 3, chronic Lyme arthritis, has been observed at the Institute of Rheumatology of the Russian Academy of Medical Sciences since 2003.

Serological diagnosis is carried out using a diagnostic test system in the format of an immunochip.

An example of a serological differential diagnosis of ICD is given for one patient.

The antigens Borrelia garinii p 100, Borrelia afzelii p 100, Borrelia garinii p41, Borrelia afzelii p41, Borrelia garinii BBK32, Borrelia afzelii BBK32, Borrelia garinii p17, Borrelia afzel, Borrelia afzel afzelii OspC, Borrelia afzelii p39, Borrelia afzelii p58, a Borrelia afzelii VlsE and VlsE Borrelia garinii, auxiliary controls (in a specific example: a mixture of anti-species antibodies to human IgG immunoglobulins and anti-species antibodies to human IgM immunoglobulins, a mixture of internal IgG immunoglobulins negative control (sorption buffer), markers of Erray boundaries). The location scheme of the immobilized spots on the errey is shown in the example of the execution of errey 2.

Insert into a Whatman slide slide holder. On top of the slide (on the labeling side), a Whatman stencil frame is put on to prevent contamination (fusion) of the samples. The immunosorbent is washed once with a working solution for washing, adding 100 μl of solution to each erray (well). Incubate on a thermal shaker with a platform rotation of 500 rpm at 37 ° C for 2-3 minutes. After incubation, the contents of the wells of the stencil frames are carefully removed without damaging the central field of the well corresponding to the location of the Herrea on the slide. Without removing the stencil, the surface of the immunosorbent is dried. Next, 90 μl of sample dilution solution is added to the immunosorbent erre. 10 μl of K + is added to one of the immunosorbent erreias, 10 μl of K- is added to the other, and 10 μl of the studied serum / plasma are added to the unoccupied herrey. When studying for the presence of antibodies in the patient's cerebrospinal fluid, the test material (cerebrospinal fluid / cerebrospinal fluid) is introduced without preliminary dilution, i.e. 100 μl The immunosorbent is closed with a lid and incubated for 30 min on a thermo-shaker with a platform rotation of 500 rpm at 37 ° C. The immunosorbent is washed with a working solution for washing, adding 100 μl of solution to each erray. Incubate on a thermal shaker with a platform rotation of 500 rpm at 37 ° C for 2-3 minutes. After that, 100 μl of the working solution for washing is introduced into each erray of the immunosorbent and incubated on a thermal shaker with a platform rotation of 500 rpm at 37 ° C for 2-3 minutes. Then, 60 μl of the conjugate working solution are added to each erosion of the immunosorbent, the immunosorbent is closed with a lid and incubated for 30 min on a thermal shaker with a platform rotation of 500 rpm at 37 ° C. The immunosorbent is washed once with a working solution for washing, adding 100 μl of solution to each erray. Incubate on a thermal shaker with a platform rotation of 500 rpm at 37 ° C for 2-3 minutes. The stencil frame is removed from the immunosorbent, the immunosorbent is rinsed with distilled water. Dry the slide. After the analysis, the slide is scanned on a multi-channel scanner for biochips, activating the channels Su 5 and Su3.

The results are recorded according to the instructions for use with the diagnostic test system in the format of an immunochip. Calculate the coefficients of positivity (CP) for all immobilized antigens. When KP> 1.1, immunoreactivity for a particular antigen is considered positive.

Fig. 3A shows a fluorescence profile of erreia showing the presence in the patient's blood serum of specific class G antibodies (IgG) to p100 antigens of Borrelia garinii and Borrelia afzelii, p58 Borrelia afzelii, p41 Borrelia afzelii, p39 Borrelia afzelii, Bbk32 Borrelia afzelii afzelii on the channel Su 5.

Figure 3B shows a profile of the same erreia, indicating the absence of class M antibodies to antigens (on the channel Su3).

As a result of the calculation, the following positivity factors (KP) were obtained.

On the channel Su 5 (the presence of IgG):

KP (p100 Borrelia garinii) - 2.2

KP (p100 Borrelia afzelii) - 7.3

KP (VlsE Borrelia garinii and Borrelia afzelii) - 0.8

KP (p58 Borrelia afzelii) - 32.4

KP (p41 Borrelia garinii) - 0.6

KP (p41 Borrelia afzelii) - 5.5

KP (p39 Borrelia afzelii) - 2.1

KP (Bbk32 Borrelia garinii) - 0.7

KP (Bbk32 Borrelia afzelii) - 48.9

KP (OspC Borrelia garinii) - 0.4

KP (OspC Borrelia afzelii) - 0.6

KP (p17 Borrelia garinii) - 0.5

KP (p17 Borrelia afzelii) - 51.1

On the channel Su3 (the presence of IgM):

KP (p100 Borrelia garinii) - 0.8

KP (p100 Borrelia afzelii) - 0.4

KP (VlsE Borrelia garinii and Borrelia afzelii) - 0.3

KP (p58 Borrelia afzelii) - 0.7

KP (p41 Borrelia garinii) - 0.6

KP (p41 Borrelia afzelii) - 0.6

KP (p39 Borrelia afzelii) - 0.4

KP (Bbk32 Borrelia garinii) - 0.6

KP (Bbk32 Borrelia afzelii) - 0.3

KP (OspC Borrelia garinii) - 0.4

KP (OspC Borrelia afzelii) - 0.5

KP (p17 Borrelia garinii) - 0.4

KP (p17 Borrelia afzelii) - 0.71

According to the interpretation of the results in the immunochip, specific class G antibodies were detected in the patient’s blood serum, which confirms the patient’s long-term infection with ICD.

Example 2

Assessment of the sensitivity and specificity of the test system in the format of an immunochip for serological diagnosis of LB.

Material and methods.

1. Immunochip for serological diagnosis of LB.

An experimental series of test systems was prepared to detect the spectrum of antibodies of classes G and M to the Borrelia garinii and Borrelia afzelii antigens. The design of one immunosorbent erreia is presented in an example of the execution of erreia 1.

The use of a single immunosorbent (slide with immobilized recombinant antigens) allows for a serological diagnosis of 12 samples of clinical material, including control sera. Analysis on a chip was carried out according to the Instructions for use (the sequence of setting and rules for interpreting the results are described in example 1).

2. Clinical material.

To assess the sensitivity in the work, serum / plasma samples from 79 patients were taken, taken up to 1 month from the moment of tick suction. In these patients, in most cases, a characteristic clinical picture was observed that was characteristic of the initial stage of tick-borne borreliosis (migratory erythema was detected in 56 patients, almost all patients noted fever). The combination of clinical and laboratory data made it possible to attribute this clinical material to stage 1 disease. Serum / plasma samples from 150 patients with disseminated and chronic stages of IKB were also studied.

To assess the specificity of the test system in the format of the immunochip, a control group was formed including 495 serums / blood plasma from individuals who did not have specific antibodies to borrelia, among which 250 samples were obtained from practically healthy donors who had never noted tick suction; 50 - from pregnant women and patients with autoimmune diseases; from patients suffering from spirochetosis of a different etiology other than borreliosis, in particular, 130 samples were obtained from patients with syphilis; 65 samples - from patients with leptospirosis.

Clinical material was provided by the Scientific Research Institute of Rheumatology, Russian Academy of Medical Sciences (Moscow), MONIKI named after Vladimirsky (Moscow), as well as by the centers of State Sanitary and Epidemiological Supervision of Moscow, St. Petersburg, Kirov, Izhevsk, Yekaterinburg, Yaroslavl and a number of other clinical bases.

The results obtained and discussion.

An analysis of the data obtained in the study of samples from patients with ICD showed that the detection of specific antibodies of classes M (IgM) among patients assigned to the first clinical stage of borreliosis reached 60.1%, and antibodies of class G - 31.6%. The detection of IgGH / or IgM confirmed the serological diagnosis of borreliosis in 87.3% of patients in this group. The detection of IgM in the group of patients with stage 2 and stage 3 LB reached 51.4%, and class G antibodies (IgG) in 88.6%. The total detection of antibodies in the chip reached 98.1%.

It should be noted that the percentage of detection of total specific immunoglobulins of both classes in patients at an early stage of the disease with the help of an immunochip is the highest among all the test systems of this purpose registered in the Russian Federation working in the ELISA and immune blot format. So, according to the results of the State comparative tests conducted in the GISK them. L.A. Tarasevich in 2003-2004, the percentage of the total detection of specific antibodies in the group of patients assigned to stage 1 LB in the ELISA test systems did not exceed 77.2% (parallel testing in test systems manufactured by Omniks LLC “Borreliosis-IFA-IgG” and “Borreliosis-IFA-IgM”), and when analyzing a sample of clinical samples from patients with UTI (2, 3 stages), the highest sensitivity - 91.3% was demonstrated by the Enzygnost Borreliosis-IgG test system + IgM ”produced by“ Dade Behring Marburg GmbH ”(Manzenyuk I.N., Manzenyuk O.Yu. in the book“ Tick-borne borreliosis ”, 2004)

Analysis of human blood serum of the above control group showed the absence of specific IgM and IgG when placed in the immunochip in 97.2%.

Our data indicate a high sensitivity, specificity of the immunochip.

The number of doubtful results in the study of the experimental group was 9.2% - in the study of the spectrum for the presence of specific anti-IgM and about 12% for the spectrum for anti-IgG. The percentage of detection of uncertain results in the chip in the analysis of the control group was slightly lower: for antibodies of class M - 5.3%, for IgG - 4%.

It was of interest to study the frequency of occurrence of antibodies to a specific antigen among a group of patients with acute form of UTI (stage 1) and patients with disseminated and chronic forms of borreliosis (stages 2 and 3). The data obtained are shown in tab. 3. The combination of clinical manifestations of LB and the analysis of the antibody pattern in the study of patients with acute and chronic stages allows us to conclude that, as an exacerbation of the disease or in the initial stage of infection, antibodies to OspC, as a rule, dominate. High levels of antibodies are also noted for p17, p41. However, during periods of exacerbation of infection or in the acute initial stage, antibodies to other antigens are much less common, especially to p100, VlsE, Bbk32.

Table.3 Identification of the spectrum of antibodies to borrelia antigens in the immunochip Antigen % detection of antibodies to antigens on the immunosorbent chip Stage IKB 2 and 3 stages of ICD IgM IgG IgM IgG p100 8.7 29.1 5.8 15.3 Vlse 2.2 26.6 2.9 17.1 p41 68.9 32.9 13.5 25.9 p58 8.9 17.7 2,4 16,4 p39 15.8 17.7 1.9 11.8 Bbk32 1,1 26.5 1,2 23.5 Ospc 68.9 34.2 67.1 22.4 p17 35.6 40.5 17.1 29.4

The study on the immunochip of clinical samples is an informative test for specialists working in the field of studying the immunopathogenesis of ICD, because allows you to simultaneously evaluate a wide range of antibodies to diagnostic / clinically significant Borrelia antigens of both classes.

Example 3

Comparative evaluation of the sensitivity of the immunochip and test systems for this purpose.

Material and methods.

1. Immunochip for serodiagnosis of ixodid tick-borne borreliosis. An experimental series of a test system was made to detect the spectrum of antibodies of classes G and M to the Borrelia garinii and Borrelia afzelii antigens. For the design of one immunosorbent erreia, see an example of the execution of erreia 1. Analysis on a chip was carried out according to the instructions for use (the sequence of setting and the rules for interpreting the results are described in example 1).

2. Test comparison systems:

- enzyme immunoassay system "Borreliosis-IFA-IgM" (LLC Omniks, St. Petersburg),

- enzyme-linked immunosorbent assay system "Borreliosis-IFA-IgG" (LLC "Omniks", St. Petersburg),

- test system in the immunoblot format “recomBlot Vorrlia _NB IgM, IgG”, manufactured by “Mikrogen”, Germany,

- test system “Borrelia LIAISON Serology Line” (DiaSorin, Italy) on a LIAISON chemiluminescent analyzer.

3. Clinical material.

In the work, serum / plasma samples from 19 patients were taken, taken within 1 month from the moment of tick suction (see Table 4, No. 1-19). The combination of clinical and laboratory data made it possible to attribute this clinical material to stage 1 disease. Also studied samples of serum / plasma from 22 patients with disseminated and chronic stages of UTI (see table 4, No. 20-41). Clinical material was provided by the Scientific Research Institute of Rheumatology, Russian Academy of Medical Sciences (Moscow), MONIKI named after Vladimirsky (Moscow), as well as by a number of centers for state sanitary and epidemiological surveillance.

The results obtained and discussion.

Testing data are presented in table 4.

Table 4. Test results for the presence of antibodies of classes G and M to borrelia in the chip №№ Serum IgM IgG Omnix DiaSorin chip Omnix DiaSorin chip one 1350-2 + + + - - + 2 1022-3 - - with me. - - +

3 855-2 + + with me. - - with me four 1194-1 - + with me. - - - 5 947-2 - - + + + + 6 954-4 - - with me. - with me + 7 1277-2 - - with me. - + + 8 989-3 - - + - - - 9 902-2 - - + - - with me 10 1042-3 - - - - - + eleven 1090-3 - - - - with me + 12 903-1 - - + - - + 13 934-3 - - + - + + fourteen 1158-1 - - - + with me + fifteen 842-3 - - + - - with me 16 945-2 - - + - - + 17 931-2 - - - + with me + eighteen 907-2 - with me. + - - - 19 1319-2 - - + - with me with me twenty one - - + + + + 21 2 + + + - + + 22 3 + + + + + + 23 four - - - - with me + 24 5 - - with me - + + 25 6 + + + - + + 26 7 - + + + + + 27 8 + + + + + + 28 9 + + + + + + 29th 10 - - + + + + thirty eleven + + + - - - 31 12 - + + - - - 32 13 + with me + - - + 33 fourteen - - + + + + 34 fifteen - - - with me + 35 16 with me + + + + + 36 17 - - - - - + 37 eighteen - + + - with me + 38 19 - + - + + + 39 twenty - - - + + + 40 21 with me with me + - with me + 41 22 - - - + - - Note: in accordance with the instructions for use and the rules for interpreting the results of a particular test system, a positive result is indicated as +, a negative result as -, a doubtful result is doubtful.

Based on our data, the sensitivity of the test system in the format of an immunochip exceeds the test system of comparison, which is most pronounced when studying clinical material from patients with the first stage of ICD. An additional study of discordant samples that yielded a positive result when tested using the immunochip and negative in test systems manufactured by Omniks LLC and DiaSorin was carried out by immune blotting using recomBlot Borrelia _NB IgM / IgG manufactured by Mikrogen, Germany. It was found that out of 13 samples containing antibodies of the IgM class that were detected using the immunochip, 5 sera according to the results in the immunoblot contained anti-IgM and 3 results were uncertain. It should be noted that 4 samples were regarded as negative, despite the presence of IgM antibodies to one VlsE or p41 antigen, because according to the rules of interpretation for the test system recomBlot Borrelia _NB IgM / IgG, the number of points was below five, which did not allow us to consider these results as positive or even doubtful.

Of the eight samples containing class G antibodies detected as a result of the analysis in the immunochip, three sera, according to the results in the immunoblot, contained anti-IgG and two results were uncertain.

An example of the execution of Herrey 1

p100 p100 Vlse Vlse p58 p41 p41 marker B.garinii B.afzelii B.garinii B.afzelii B.afzelii B.garinii B.afzelii p100 p100 VIsE VIsE p58 p41 p41 marker B.garinii B.afzelii B.garinii B.afzelii B.afzelii B.garinii B.afzelii p39 VVK32 VVK32 Ospc Ospc p17 p17 marker B.afzelii B.garinii B.afzelii B.garinii B.afzelii B.garinii B.afzelii p39 VVK32 VVK32 Ospc Ospc p17 p17 marker B.afzelii B.garinii B.afzelii B.garinii B.afzelii B.garinii B.afzelii Human IgG concentration 1 Human IgG, concentration 2 Human IgG concentration 3 Human IgG concentration 4 Human IgG concentration 5 Human IgG concentration 6 sorption buffer marker Human IgG, concentration 1 Human IgG, concentration 2 Human IgG, concentration 3 Human IgG, concentration 4 Human IgG concentration 5 Human IgG concentration 6 sorption buffer marker Human IgM, concentration 1 Human IgM, concentration 2 Human IgM, concentration 3 Human IgM, concentration 4 Human IgM concentration 5 Human IgM concentration 6 sorption buffer marker Human IgM, concentration 1 Human IgM, concentration 2 Human IgM, concentration 3 Human IgM, concentration 4 Human IgM concentration 5 Human IgM concentration 6 sorption buffer marker marker marker marker marker marker marker marker marker

An example of the execution of Herrey 2

marker p100 B.gar p100 B.afz Vlse marker marker p100 B.gar p100 B.afz Vlse marker p58 B.afz p41 B.gar p41 B.afz p39 B.afz VVK32 B.gar p58 B.afz p41 B.gar p41 B.afz p39 B.afz VVK32 B.gar VVK32 B.afz OspC B.gar OspC B.afz p17 B.gar p17 B.afz VVK32 B.afz OspC B.gar OspC B.afz p17 B.gar p17 B.afz marker negative control (sorting buffer) anti-human IgG + anti-human IgG Human IgM + human IgG marker marker negative control (sorption buffer) anti IgG Human IgM + human IgG marker

Thus, the proposed diagnostic test system in the form of an immunochip can improve the efficiency of serological analysis of ICD, because the system proposed in accordance with the present invention can detect a wide range of antibodies in human serum / plasma and / or cerebrospinal / synovial fluid and at the same time differentiates them by classes G and M on one immunosorbent.

The proposed method of serological differential diagnosis using a diagnostic test system in the format of an immunochip is highly sensitive and specific, easy to perform, eliminates the subjectivity of the assessment, thanks to the automated interpretation of the results. When conducting analysis using the proposed diagnostic test system in the format of an immunochip, a minimum volume of the test material is required.

Claims (6)

1. Diagnostic test system in the format of an immunochip for serological diagnosis of tick-borne ixodic borreliosis, including immunosorbent in the form of a slide, divided into Erreys, each of which is designed to analyze one sample, or in the form of an immunological tablet, each well of which is designed to analyze one sample, with slots containing diagnostically significant antigens immobilized within the boundaries of each erreia or well of the tablet / synthetic peptides belonging to the complex Borrelia burgdorferi sensu lato, as well as auxiliary control spots for standardizing the analysis on the immunochip and verifying the validity of the test system, a conjugate consisting of a mixture of secondary antibodies to human IgG and antibodies to human IgM modified with two different spectral characteristics of fluorophore dyes. 2. A diagnostic test system in the format of an immunochip for serological diagnosis of tick-borne tick-borne borreliosis according to claim 1, additionally containing reagents necessary for conducting and validating the analysis. 3. The diagnostic test system in the format of an immunochip for serological diagnosis of ixodid tick-borne borreliosis according to claim 1, where each herrey slide or well of the tablet contains a combination of separately immobilized slots, each of which contains at least one diagnostically significant recombinant antigen / synthetic peptide selected from the group: Borrelia garinii p 100, Borrelia afzelii p100, Borrelia garinii p41, Borrelia afzelii p41, Borrelia garinii BBK32, Borrelia afzelii BBK32, Borrelia garinii p17, Borrelia afzelii p17, Borrelia afelpelzelzpel, Borrelia afelpelzelzpz p58, Borrelia afzelii VlsE, Borrelia garinii VlsE. 4. The diagnostic test system in the format of an immunochip for serological diagnosis of tick-borne ixodic borreliosis according to claim 1, where the auxiliary control spots within each erythema of a slide or well of a tablet for standardizing analysis on an immunochip can be represented by at least one of the following options: - a number of spots containing the same concentration of any protein / peptide modified with fluorophore dyes that are insignificant for serological diagnosis of ixodid tick-borne borreliosis, similar to those used in the conjugate of the test system, - a number of spots containing different concentrations of human IgG immunoglobulin, - a number of spots containing different concentrations of human IgM immunoglobulin, - a number of spots containing various concentrations of antibodies to human IgG, - a number of spots containing various concentrations of antibodies to human IgM, - a negative control within each errey is a sorption buffer that does not contain antigen or another protein. 5. The diagnostic test system in the format of an immunochip for serological diagnosis of tick-borne ixodic borreliosis according to claim 1, where the immunosorbent is a slide in the form of a glass or plastic base with a chemically modified surface or coated with nitrocellulose. 6. The diagnostic test system in the format of an immunochip for serological diagnosis of tick-borne ixodic borreliosis according to claim 2, where, as reagents necessary for conducting and validating the analysis, the diagnostic test system contains a solution for diluting control and test samples, a solution for diluting the conjugate, solution for washing the immunosorbent, a positive control sample (K +), a negative control sample (K-).

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