Diagnostic test system in immunochip format and differential serum diagnostics of syphilis

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns development of a diagnostic test system in immunochip format and a method of simultaneous and differential detection of reaginic antibodies and antibody spectrum to diagnostically significant mmunologically relevant proteins Treponema pallidum of G (IgG) and M (IgM) classes. The diagnostic test system in immunochip format for differential serum diagnostics of syphilis consists of an immunosorbent with separately immobilised antigens Treponema pallidum Tp15, Tp17, TmpA, Tp47, conjugate and reactants required to detect an antigen-antibody complex. Cardiolipin is additionally immobilised on the immunosorbent; antigens Treponema pallidum and cardiolipin are immobilised at least, in two repetitions, and conjugate consists of mixed antispecies human IgG antibodies and human IgM antibodies modified by two phosphors with different spectral characteristics. On the immunosorbent, there can be additionally immobilised at least, one antigen and/or peptide Treponema pallidum specified e.g. of Tp39, Tp41, Tp42, Tp44.5, Tp92, Tp0453 at least in two repetitions. Cardiolipin can represent an oxidised cardiolipin derivative bounded with protein. The differential serum syphilis diagnostic technique with using the diagnostic test system in immunochip format involves application of human IgM modified by two phosphors with different spectral characteristics. On the immunosorbent, there can be additionally immobilised at least one antigen and/or peptide Treponema pallidum, specified e.g. of Tp39, Tp41, Tp42, Tp44.5, Tp92, Tp0453 at least in two repetitions. Cardiolipin can represent an oxidised cardiolipin derivative bounded with protein. The differential serum syphilis diagnostic technique with using the diagnostic test system in immunochip format implying that a cultivation solution for check and test samples is introduced on the immunosorbent with separately immobilised antigens Treponema pallidum and cardiolipin; the check and test samples are introduced; the prepared mixture is incubated at temperature 20-42°C for 15-60 min to prepare the antigen-antibody complex; the immunosorbent is rinsed; then a conjugate solution of mixed human IgG antibodies and human IgM antibodies modified by two phosphors with different spectral characteristics is introduced on the immunosorbent; it is followed with incubation at temperature 20-42°C for 15-60 min; the immunosorbent is rinsed, dried to detect the prepared antigen-antibody complex.

EFFECT: improved diagnostic accuracy.

20 cl, 5 dwg, 2 ex

 

The invention relates to the field of clinical laboratory diagnostics, biotechnology and medicine and relates to a diagnostic test system in the format immunochip (biological chip and method for the diagnosis of syphilis using this immunochip allow for the simultaneous and differential identify reginavia and treponemataceae antibodies (antibodies to Treponema pallidum) different classes.

Over the last decade, the Russian Federation has experienced an increase in the proportion of early latent syphilis, representing epidemiological danger, and late forms, leading to severe lesions of the nervous system and internal organs [Collection of key indicators providing dermatological care to the population (statistics), 1997; Resources and activities of STI institutions. The incidence in the 2003-2004, 2004-2005, 2005-2006 (statistics)].

In connection with the peculiarities of the clinical course of syphilis necessary complexity laboratory studies using non-treponemal and treponemal tests. Serological diagnosis of syphilis infection in the Russian Federation is regulated by the decree of the Ministry of health of the Russian Federation No. 87 dated 26.03.2001, "On improving the serological diagnosis of syphilis", which for the diagnosis of syphilis should conduct is their analysis using a non-treponemal tests, and additional analysis using treponemal tests.

Non-treponemal tests that determine reginavia antibodies heterophile antibodies generated during tissue damage due to invasion of syphilis Treponema pallidum, or to lipids of the cell wall Treponema pallidum - are used to formulate a preliminary serological diagnosis. Main tests in this group are the RMP - reaction microprecipitation, RSCC - reaction of complement fixation with cardiolipin antigen (mixture of cardiolipin, cholesterol, lecithin) or Wasserman, VDRL - test Research laboratory sexually transmitted diseases, RPR - rapid plasmalogens test and others. Listed non-treponemal tests are based on the definition in the studied material of antibodies to cardiolipin. The advantages of non-treponemal tests are simplicity of implementation and low cost. Semiquantitative analysis with determination of the titer ragenovich antibodies allows their use in differentiating relapse from reinfection and in assessing the effectiveness of treatment. However reginavia antibodies are not always specific in relation to the syphilis pathogen, and can temporarily be detected in the serum of people with systemic diseases parenchymal organs after immunization during pregnancy, the elderly and oncab is selected [EcoClassic et al. Guidelines for the laboratory diagnosis of syphilis in Eastern Europe // journal of dermatology and venereology, 2008. No. 5. - P.87-96]. Evaluation of the results of the analysis using non-treponemal tests usually carried out visually.

Treponemal tests that define specific antibodies to Treponema pallidum, are more specific. The most common treponemal tests are ELISA - enzyme-linked immunosorbent assay, TPHA - Treponema pallidum haemagglutination assay, RIBT - reaction immobilization pale treponemes, REEF - reaction immunofluorescence assay, IB - immune cell type.

Enzyme-linked immunosorbent assay (ELISA) allows you to define one hole immunological tablet specific antibodies or to only one antigen or mixture of antigens of Treponema pallidum. For differential identification of the spectrum of antibodies to syphilis must be posted diagnostically relevant antigens of Treponema pallidum by individual wells. To achieve the separate detection of antibody classes (IgG - immunoglobulin G, IgM - immunoglobulin M) use individule compared to IgG and IgM man made in different wells of immunoplate. Often for this purpose we use two test systems intended for the detection separately antibodies of class G antibodies of class M, which significantly increases the time of carrying out the ia analysis, its cost, requires increasing the volume of the analyzed biological material.

Immune blotting (IB), despite separate immobilization of antigens and, therefore, separate detection of antibodies to syphilis, also does not allow one immunosorbent - stripe (membrane) - differential to determine whether a detected treponemataceae antibodies of multiple classes. One clinical sample for the presence of antibodies of class G and M there is a need to use 2 strips for the separate application of two antivitamin conjugates. One strip is used for detection with conjugate anti-human IgG, and the other with conjugate anti-human IgM. The disadvantages of this test should include the subjectivity of the interpretation of the results when the visual assessment, the high cost sets.

Test the reaction of immobilization pale treponemes (REBT) not suitable for the diagnosis of early stages of infection. The reaction of immobilization pale treponemes becomes positive not previously 3-6 weeks from the moment of infection. This test is complicated, time consuming and expensive, requiring highly qualified staff and the availability of the vivarium for maintaining laboratory strain pale treponemes.

Test reactions immunofluorescence assay (RIF) is simple in formulation, sensitive at all stages of infection, However, for setting this test also requires live pale Treponema. In addition, this test may give false positive results in long execution time-consuming, expensive and requires highly skilled personnel.

Test passive haemagglutination (GPA) has a low sensitivity in primary syphilis.

Known diagnostic device to determine syphilis, made in the form of a plastic tablet for immunofermentnogo analysis with holes on the inner side of which is a substance that reacts with antibodies to syphilis [a patent for useful model No. 7874, IPC AC 39/12]. In the composition of the material includes several different peptide fragments of protein antigens of Treponema pallidum. However, this device does not allow to identify reginavia antibodies, as well as to determine simultaneously antibodies of different classes.

Known protein chip for testing infections in the serum, including antibodies to the same antigen syphilis [application PRC No. 1373365, IPC G01N 33/68; G01N 33/569; G01N 33/576]. However, this chip is not possible to determine reginavia antibodies, as well as to determine a spectrum of antibodies to Treponema pallidum and to simultaneously identify antibodies of different classes.

A known test system in the form of a biological chip, based on the reactions of the interaction of the antibody with the antigen and its application [Patent of Russia № 2316000, IPC G01N 33/53; G01N 33/543; G01N 33/56]. However, this test system tacrine allows you to simultaneously identify reginavia antibodies and range treponemataceae antibodies. In addition, the biochip of this test system includes only one antigen syphilis.

Thus, none of the currently known diagnostic tests simultaneously and differentially detect reginavia antibodies and range treponemataceae antibodies (specific antibodies to Treponema pallidum), i.e. to combine in one of two different test: treponemal and non-treponemal. In addition, none of the known diagnostic tests does not allow simultaneously and differentially to identify antibodies of different classes, important in the diagnosis of syphilis - IgG and human IgM in serum/plasma human blood or cerebrospinal fluid.

Closest to the present invention is a kit for detection of antibodies to pale the spirochete Treponema pallidum, which contains the reagents necessary for the detection of complex antigen-antibody, and adsorbed on the solid phase recombinant polypeptide antigens of Treponema pallidum 15 kDa, 17 kDa, Try and 47 kDa, and these antigens are adsorbed on the solid phase in the total mixture, or separately, or in the total mixture and one antigen adsorbed separately and, in addition, each of the other pairs absorbed together with him [the Patent of Russia № 2103362, IPC 12N 15/31, SC 14/20]. This invention is selected as a prototype diagnostic test systems in the format of immunos the PA.

The technical task of the present invention is to develop a diagnostic test systems in the format of immunochip and method for the simultaneous and differential detection ragenovich antibodies and the range of antibody diagnostically significant immunogenic proteins of Treponema pallidum class G (IgG) and M (IgM) for high-precision and highly specific diagnosis of syphilis in the early stages of the disease.

The technical problem is achieved by the fact that the diagnostic test system in the format immunochip for serological differential diagnosis of syphilis consists immunosorbent assay with immobilized thereon separately antigens of Treponema pallidum Tr, Tr, Tmra, Tr, conjugate and reagents required for detecting the complex of antigen-antibody. On the immunosorbent optionally immobilized cardiolipin, and the antigens of Treponema pallidum and immobilized cardiolipin at least two repeats, and the conjugate consists of a mixture of antivitamin antibodies to human IgG and IgM antibodies to human modified in two different spectral characteristics of the fluorophores.

On the immunosorbent and additionally separately immobilized at least one antigen and/or peptide Treponema pallidum, selected, for example, from Tr, Tr, Tr, Tr, Tr, Tr at least two repetitions.

Cardiolipin is an oxidized derivatives of the same cardiolipin, United with protein.

On the immunosorbent optionally immobilized auxiliary controls at least two repetitions.

Auxiliary controls are antibodies to the immunoglobulin IgG and/or antibodies to immunoglobulin IgM and/or immunoglobulin IgG and/or immunoglobulin IgM and/or internal negative control and/or markers of the boundaries of Arreau.

Immunosorbent is a slide in the form of a glass or plastic substrate with surface modified or coated with nitrocellulose.

Immunosorbent made in the form of a slide into array, each of which is designed to analyze a single sample.

Immunosorbent can be a polystyrene tablet, each hole which is designed for analysis of one sample.

As the reagents necessary for the detection of complex antigen-antibody diagnostic test system contains a diluent specimens and controls, diluent conjugate, the rinse solution immunosorbent assay, a positive control sample (K+), a negative control sample ( -).

The way serological differential diagnosis of syphilis with diagnostic test systems in the format of immunochip according to claim 1, comprising depositing on immune rent solution for cultivation specimens and controls, making specimens and controls, followed by incubation at a temperature of 20-42°C for 15-60 min for the formation of complex antigen-antibody, immunosorbent washed, next to immunosorbent contribute conjugate solution and carry out the incubation at a temperature of 20-42°C for 15-60 min, immunosorbent washed, dried, followed by detection of the formed complexes antigen-antibody and identify reginavia and treponemataceae antibodies of class G (IgG) and/or M (IgM), based on estimates of signal intensity of fluorescence upon activation of the respective channels of the multichannel fluorescence scanner.

On immunosorbent additionally separately immobilized at least one antigen and/or peptide Treponema pallidum selected from CR, Tr, Tr, Tr, Tr, Tr at least two repetitions. Cardiolipin is an oxidized derivative of cardiolipin, United with protein.

Control samples represent a positive control sample (+) and negative control sample ( -).

On the immunosorbent and additionally immobilizer auxiliary controls at least two repetitions.

Auxiliary controls are antibodies to the immunoglobulin IgG and/or antibodies to immunoglobulin IgM and/or immunoglobulin IgG brow the ESA and/or immunoglobulin IgM and/or internal negative control and/or markers of the boundaries of Arreau.

Immunosorbent is a slide in the form of a glass or plastic substrate with surface modified or coated with nitrocellulose.

Immunosorbent made in the form of a slide into array, each of which is designed to analyze a single sample.

Immunosorbent can be a polystyrene tablet, each hole which is designed for analysis of one sample.

Identifying ragenovich antibodies is carried out in the location of cardiolipin on the immunosorbent.

Identifying treponemataceae antibodies is carried out in places of localization of antigens of Treponema pallidum on the immunosorbent.

Diagnostic test system contains immunosorbent with immobilarie it separately diagnostically relevant antigens and/or peptides Treponema pallidum and cardiolipin, conjugate and reagents required for detecting the complex of antigen-antibody. On the immunosorbent can optionally be immobilized internal controls.

Reagents for detection of complex antigen-antibody are:

the diluent study and control samples (e.g., 1% solution of casein and 2% bovine serum albumin in phosphate-buffered saline),

the diluent conjugate (e.g., 1% solution of casein in phosphate-buffered saline with 0.1% tween-20),

- rest the R for washing immunosorbent assay (for example, phosphate-saline buffer containing 0.1% tween-20),

- positive control sample (+) - inactivated by heating with a pool of sera or plasma from patients with syphilis, containing reginavia and treponemataceae antibody classes G and M,

negative control sample ( -) - inactivated by heating with a pool of sera or plasma from healthy donors, not containing reginavia and treponemataceae antibody classes G and M

The most economically feasible conducting simultaneous group analysis of the samples using immunosorbent assay hosting from 12 to 16 areev (identical zones) with immobilized important antigens of Treponema pallidum, cardiolipin and auxiliary controls. Each array is used for production of the same subject or control sample. When performing the analysis on one array make a positive control sample of the test system (+) to study the validity of the test system. Another array take a negative control sample, the test-system (K-) for subsequent data interpretation immunoassay. The remaining unoccupied array make the analyzed samples.

Often the solid phase for immunocyto serve microscope slides of a width of 25 ± 1 mm, length 75+1 mm and thickness of 1 mm with a modified surface is thew (may contain epoxy-, aldehyde, amino groups, polymers, hydrogels, and the like), or coated with nitrocellulose. For exceptions merge samples array share a special device - under-sealants. There are commercially available frame and slide holders, for example, the company "Whatman, USA, separating array in the analysis.

Print technology allows the application of biological substances are also at the bottom of the wells of polystyrene tablet for ELISA, which makes possible the use of the tablet as immunosorbent assay, and one hole of the tablet corresponds to one array and is intended for analysis of one sample.

With advanced print settings - plotters - diagnostically relevant antigens of Treponema pallidum, cardiolipin, auxiliary controls immobilized (adsorb) to the surface of the solid phase in the form of individual dots or spots ranging in size from 100 to 500 microns. To avoid errors when printing (pass printing) or in the process of analysis immunochip each antigen cardiolipin and auxiliary controls immobilized separately at least two times. When analyzing the test results of the samples in the calculated average of the spot value of the fluorescence signal. The print quality is additionally controlled by introducing into the solution sorbed on the solid phase antigens, ka is diolefin and auxiliary controls any nonspecific for the diagnosis of syphilis protein, for example, BSA (bovine serum albumin), modified fluorophores with different spectral characteristics from those used in the conjugate diagnostic test systems. For example, the introduction of BSA modified fluorophores FITC, allows for the appropriate channel (in particular, FITC at excitation wavelength is 488 nm, 512 nm emission) when scanning multi-channel scanner for biochips to register fluorescent spots, according to their localization on arree. This is very useful when the overlay mesh," in processing of the results of the analysis using specialized software that came with the scanner, with the aim of correct reading of signal intensity of fluorescence within each spot and background on array outside spots.

Auxiliary controls are:

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

immunoglobulin IgG and/or immunoglobulin IgM person serve to control the introduction of conjugate solution in array;

- antibodies to immunoglobulin IgG and/or antibodies to immunoglobulin IgM person serve to control the introduction of the sample in array;

markers of the boundaries of Arreau that immobilized boundaries of each area allow after analysis to visually observe the location of arree and to control the overlay grid when using the program scoring results.

As markers of the boundaries of Arreau often immobilised any nonspecific for the diagnosis of syphilis protein, such as BSA, modified one of the fluorophores.

The invention is illustrated in the drawings.

Figure 1 - version immunosorbent assay diagnostic test kits for serological differential diagnosis of syphilis in the form of a slide containing 12 Arreau.

Figure 2 is an enlarged scale array.

Figure 3 - an example of execution of array.

Figure 4 - example of a fluorescence image profiles areev on different channels for a patient with a positive serologic diagnosis of syphilis.

Figure 5 - example of a fluorescence image profiles areev on different channels for a patient with negative serologic diagnosis of syphilis.

Immunosorbent is a slide 1 (Figure 1) in the form of a glass or plastic substrate with a modified surface, which is divided into array 2 located at the same distance from each other. Border 3 areev 2 to prevent mergers samples provide overlay frame (frame not shown). Array 2 (2) contain the 4 spots in the form of individual pixels.

On immunosorbent separately immobilized diagnostically relevant antigens and/or peptides Treponema pallidum. Most often when syphilis detected antibodies to the R17. The use of this protein provides maximum sensitivity analysis. Protein Tr is a sensitive marker IgM response in congenital infection. Detection of antibodies simultaneously on Tr and Tr helps differentiate syphilis with suspected Lyme disease (tick-borne borreliosis). It is noted that in the course of treatment in the first place, there is a decrease in the titer of antibodies to TMR. Antigens Tr, Tr, Tr, Tmra - recognized and a minimum set of diagnostically important proteins of Treponema pallidum for serodiagnosis of syphilis. The broader spectrum treponemataceae antibodies allows not only to identify early stage of infection, but also to confirm serological diagnosis. Analysis of the primary structure of the genome of Treponema pallidum helped to identify additional antigens for the diagnosis of syphilis. In particular, protein Tr can be used as a highly specific marker Treponema pallidum to determine the early stage of infection with syphilis. Diagnostically important proteins are other antigens, for example CR, Tr, Tr, Tr, Tr. Additional introduction part immunosorbent assay of these proteins to the set of the four most used protein Tr, Tr, Tr, Tmra increase the sensitivity of a diagnostic test.

In addition to antigens and/or peptides Treponema pallidum, immunosor the NT diagnostic test systems in the format of immunochip includes cardiolipin to identify ragenovich antibodies.

Order irreversible adsorption of the drug on the surface of the solid phase and increase shelf life immunosorbent assay without reducing the antigenic properties of cardiolipin, previously conducted chemical modification of the drug to obtain the oxidized derivative cardiolipin connected with any that are not important for the diagnosis of syphilis protein (e.g., BSA).

With the purpose of determination of antibodies of different classes on the same immunosorbent assay used conjugate, which is a mixture of antivitamin antibodies to human IgG and IgM person who modified respectively different spectral characteristics of the fluorophores. Antibodies to human IgG modified, for example, a fluorophore So, and antibodies to human IgM modified, for example, a fluorophore SS3. In the scanning process when you activate the corresponding channel fluorescence scanner becomes possible differentiation of antibodies of different classes. For this program the scanner type used in the conjugate of the fluorophores, and the scanner automatically activates the appropriate channels.

Differential detection of antibodies to classes is important in the diagnosis of syphilis. First after infection, antibodies of class M (IgM)detected in defined quantities in the second week and reaching the maximum the Oh concentration in the blood at 6-9 week. Disease relapse or reinfection lead to a new increase in the concentration of IgM, and in case of recurrence of the antibodies of this class are often produced even in higher concentrations than in primary infection. After effective treatment, the concentration of IgM decreases rapidly, and usually 3-9 months they no longer determined in the serum. Antibodies of class G (IgG) in defined quantities appear in the blood after 3-4 weeks from the time of infection. Their concentration increases at week 6 begins to dominate over the concentration of IgM, and, reaching the maximum, is maintained at a certain level for a long time. After effective treatment, the concentration of immunoglobulin is gradually decreasing, but this is much slower than in the case of IgM.

Separate identification of the spectrum of antibodies to Treponema pallidum is important to confirm the setting serological diagnosis of syphilis. It is proved that the detection of antibodies to two or more antigens indicates a positive serological diagnosis of syphilis. At the same time the detection of antibodies to only one of the major antigens of the causative agent of syphilis is not possible to consider the result of a serological assay as positive. In this case, it is treated as uncertain, requiring further monitoring is inimicos antibody production. The dynamics of the developments of antibodies to various diagnostically important proteins allows clinicians to predict the course of illness.

Method for the serological diagnosis of syphilis with diagnostic test systems in the form of immunochip as follows.

On immunosorbent with pre-immobilized separately important antigens of Treponema pallidum Tr, Tr, Tmra, Tr, and cardiolipin, contribute diluent specimens and controls, then make the control and test samples (preferred destination breeding contributed samples to the diluent 1:10). Then carry out incubation of the mixture at a temperature of 20-42°C for 15-60 min (preferably using thermocamera with the rotation of the platform 500 revolutions per minute) for the formation of complex antigen-antibody. Further immunosorbent, in order to remove unreacted components, washed at least one-two times a rinse solution immunosorbent assay, for example, 1 × phosphate-saline buffer (pH 7.2) supplemented with 0.1% tween-20.

On immunosorbent contribute conjugate solution of a mixture of anti-IgG and anti-human IgM, modifitsirovanniy[ various spectral characteristics of the fluorophores, previously prepared from the conjugate and the diluent conjugate. P is avodat incubation at a temperature of 20-42°C for 15-60 minutes For best results, the incubation is carried out with the use of thermocamera with the rotation of the platform 500 revolutions per minute After that immunosorbent wash working solution for washing immunosorbent assay and dried for subsequent detection results of the analysis.

On immunosorbent can additionally be separately immobilized at least one antigen and/or peptide Treponema pallidum, selected, for example, from the group CR, Tr, Tr, Tr, Tr, Tr, and auxiliary controls (antibodies to immunoglobulin IgG and/or antibodies to immunoglobulin IgM and/or immunoglobulin IgG and/or immunoglobulin IgM and/or internal negative control and/or markers of the boundaries of Arreau). All antigens and/or peptides Treponema pallidum, cardiolipin and auxiliary controls are immobilized, at least two repetitions. In the private version using oxidized derivative cardiolipin connected with any that are not important for the diagnosis of syphilis protein (e.g., BSA).

The detection of the formed complex antigen-antibody carried out using a multichannel fluorescence scanner when you activate the corresponding channel. Processing the scan results carried out according to the instructions to use the software that came with the scanner. Identification of real the new antibodies conduct, based on estimates of signal intensity of fluorescence in the location of cardiolipin on the immunosorbent. Identifying treponemataceae antibodies are conducted based on assessment of signal intensity of fluorescence signals in places of localization of antigens of Treponema pallidum on the immunosorbent.

For each immobilized cardiolipin antigen and calculate the ratios of fluorescence, representing the ratio of the mean fluorescence values of the corresponding spots (minus background) to the background of area. Background of area consider the average value of the fluorescence of area outside spots. Next, calculate the critical value of the ratio of fluorescence (cut off) for each of the antigens of Treponema pallidum and cardiolipin. The critical value of the ratio of fluorescence for each antigen and cardiolipin is calculated by multiplying the ratio of fluorescence in arree made with a negative control sample ( -) on the alpha value (set by the manufacturer of the biochip for each series of chips by statistical analysis). Calculate the odds of positivity for each sample for all immobilized antigens and cardiolipin. The ratio of positivity is the ratio of the coefficients of the fluorescence to the critical level of coefficientpossible (cut off). The value of the coefficients is positive conclusions about the presence or absence of antibodies to immobilized antigens and cardiolipin.

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

on one specific channel, for example Su (determination of antibodies of class G) within each area should fluoresce spots, corresponding immobilized immunoglobulin IgG person. The fluorescence signal of these spots should be not less than three times above the level of fluorescence background on arree,

on the second specific channel, for example SS3 (antibody class M) within each of array should fluoresce spots, corresponding immobilized immunoglobulin IgM person. The fluorescence signal of these spots should be not less than three times above the level of fluorescence background on arree,

the level of fluorescence of the spots corresponding to the localization of the internal negative control (sorption buffer) must not exceed the level of fluorescence background of area more than two times,

the level of fluorescence of all of the spots corresponding to the localization of antigens of Treponema pallidum and cardiolipin, on specific channels A and SS3 in arree made with a negative control sample ( -) test system should not exceed the level of the HB fluorescence background on arree more than twice.

Next, perform a differential diagnosis of syphilis.

The sample is considered negative for the presence of specific and ragenovich antibodies of class G (IgG) to syphilis, if the channel So for all antigens and cardiolipin identified coefficients positivity of less than 1.

The sample is considered positive for the presence of specific antibodies of class G (IgG) to syphilis, if the channel So to two or more antigens of Treponema pallidum defined coefficients are positive, equal to or more than 1.

The sample is considered positive for the presence of ragenovich antibodies of class G (IgG), if the channel A in the location of the cardiolipin identified coefficients are positive, equal to or more than 1.

The sample is considered indeterminate for the presence of specific antibodies of class G to syphilis, if the channel So coefficients are positive with values equal to or more than 1, is defined to only one antigen.

The sample is considered positive for the presence of specific antibodies of class M (IgM) to syphilis, if the channel SS3 to two or more antigens identified coefficients are positive, equal to or more than 1.

The sample is considered negative for the presence of ragenovich antibodies of class M (IgM), if the channel SS3 in the location of the cardiolipin identified coefficients positivity of less than 1.

Will obrasatsia indeterminate for the presence of specific antibodies of class M (IgM) to syphilis, if the channel SS3 to the same antigen revealed the odds of positivity, greater than or equal to 1.

The sample is considered negative for the presence of specific and ragenovich antibody class M (IgM) to syphilis, if the channel SS3 for all antigens and cardiolipin identified factors positivity of less than 1.

Method for the serological diagnosis of syphilis using the test system in the format immunochip characterized examples.

Example 1.

Differential serological diagnosis of syphilis in the analysis of blood serum of the patient P.

An example of performing serological differential diagnosis of syphilis are given for one patient.

Perform production analysis using the diagnostic test system in the format immunochip.

Diagnostic test-system in the format immunochip includes immunosorbent, conjugate and reagents. Immunosorbent predstavljaet a glass slide by CEL Associates, Inc, USA, with a modified aldehyde groups to the surface on which separately immobilized in the form of spots antigens of Treponema pallidum Tr, Tr, Tmra, Tr, Tr, Tr, Tr, cardiolipin, auxiliary controls (a mixture of antibodies to immunoglobulins IgG and IgM man, a mixture of immunoglobulins IgG and IgM human internal negative control, markers of the boundaries of Arreau). The schema location is of immobilized spots on arree shown in Figure 3.

The conjugate is a mixture of two antivitamin antibodies to immunoglobulins IgG and IgM person.

Reagents are:

the diluent specimens and controls (1% solution of casein and 2% bovine serum albumin in phosphate-buffered saline),

the diluent conjugate (1% solution of casein in phosphate-buffered saline with 0.1% tween-20),

- rinse solution immunosorbent assay (phosphate-saline buffer containing 0.1% tween-20),

- positive control sample (+) - inactivated by heating with a pool of sera or plasma from patients with syphilis, containing reginavia and treponemataceae antibody classes G and M,

negative control sample ( -) - inactivated by heating with a pool of sera or plasma from healthy donors, not containing reginavia and treponemataceae antibody classes G and M

Insert into the frame-holder for slides firms Whatman slide. On the top of the slide (on the side of the label is put on a frame-stencil company "Whatman, USA, to prevent contamination (merger) of the samples. Washed immunosorbent once a working solution for cleaning, making each array (well) in 100 μl of solution. Incubated in thermoshakers with the rotation of the platform 500 rpm at 37°C for 2-3 minutes After incubation, the contents and the holes of the under-stencil carefully Braut, without damaging the Central hole corresponding to the location of area on the slide. Without removing the stencil, dry surface immunosorbent assay. Later in array immunosorbent assay contribute 90 ál of sample diluent. In one of Arreau immunosorbent assay contribute 10 ál To+make another 10 μl of K-, unoccupied array make a 10 µl sample of the blood serum of the patient. Immunosorbent covered with a lid and incubated for 30 min at thermoshakers with the rotation of the platform 500 rpm at 37°C. Washed immunosorbent working solution for washing, making each array 100 μl of a solution. Incubated in thermoshakers with the rotation of the platform 500 rpm at 37°C for 2-3 minutes then make 100 ál of working solution for cleaning in each array immunosorbent assay and incubated in thermoshakers with the rotation of the platform 500 rpm at 37°C for 2-3 minutes Then every array immunosorbent assay Wasat 60 ál of working strength conjugate, close immunosorbent with a lid and incubated for 30 min at thermoshakers with the rotation of the platform 500 rpm at 37°C. Washed immunosorbent once a working solution for cleaning, making each array 100 ál solution. Incubated in thermoshakers with the rotation of the platform 500 rpm at 37°C for 2-3 minutes Remove from immunosorbent frame-stencil, immunosorbent rinsed with distilled water. Dried slides analysis slide scans on multi-channel scanner for biochips activating channels A and SS3.

Conduct profitability analysis according to the instructions for use for diagnostic test systems in the format of immunochip. For each antigen and cardiolipin calculate the ratio of fluorescence. The critical value of the ratio of fluorescence for each antigen and cardiolipin is calculated by multiplying the ratio of fluorescence in arree made with a negative control sample () on the value of alpha equal to 2. Calculate the coefficients of ositively (KP) for all immobilized antigens and cardiolipin.

On Figa shows the fluorescence profile of area, demonstrating the presence of specific antibodies of class G (IgG) to antigens Tr, Tr, Tmra, Tr, Tr, Tr, Tr channel So.

On Figb demonstrated a profile of the same area channel SS3, indicating the presence of ragenovich antibodies of class M to cardiolipin.

In the calculation were obtained the following coefficients of positivity (CP).

Channel Su (presence of IgG):

- KP (Tr)=17.7

- KP (Tr)=22.8

- KP (Tmra)=20.5

- KP (Tr)=23.7

- KP (Tr)=22.0

- KP (Tr)=25.1

- KP (Tr)=19.9

- KP (cardiolipin)=0.5

Channel SS3 (presence of IgM):

- KP (Tr)=0.4

- KP (Tr)=0.5

- KP (Tmra)=0.4

- KP (Tr)=0.5

- KP (Tr)=0.4

- KP (Tr)=0.4

- KP (Tr)=0.6

- KP (cardiolipin)=17.8

In the result of the verification is to be placed serological analysis using immunochip identified treponemataceae antibodies of class G (KP for antigens Tr, Tr, Tmra, Tr, Tr, Tr, Tr greater than 1) and reginavia antibodies of class M (KP to cardiolipin greater than 1), indicating that infection of the patient P. syphilis.

Example 2.

Analysis of the blood serum of the patient M with diagnostic test systems in the format of immunochip.

The formulation of the analysis was similar to the scheme described in example 1.

On Figa shows an example of the fluorescent profile of area on different channels showing negative results for the presence of treponemataceae and ragenovich antibodies of class g

On FIGU shows an example of the fluorescent profile of area on different channels showing negative results for the presence of antibodies of class M

In the calculation were obtained the following coefficients of positivity (CP).

Channel Su (presence of IgG):

- KP (Tr)=0.3

- KP (Tr)=0.4

- KP (Tmra)=0.5

- KP (Tr)=0.5

- KP (Tr)=0.6

- KP (Tr)=0.5

- KP (CR)=0.7

- KP (cardiolipin)=0.5

Channel SS3 (presence of IgM):

- KP (Tr)=0.3

- KP (Tr)=0.6

- KP (Tmra)=0.4

- KP (CR)=0.7

- KP (Tr)=0.4

- KP (CR)=0.7

- KP (Tr)=0.6

- KP (cardiolipin)=0.2

As a result of a serological assay using immunochip not identified treponemataceae and reginavia antibodies of class G and M (KP for antigens Tr, Tr, Tmra, Tr, Tr, Tr, Tr, to Gilpin less than 1).

The proposed diagnostic system in the form of immunochip can improve the effectiveness of the serological analysis of syphilis, because the system combines two types of tests (non-treponemal and treponemal) in the same format, identifies a wide range of antibodies in serum/plasma human blood or cerebrospinal fluid and simultaneously differentiates them according to the classes of G and M on the same immunosorbent assay.

The proposed method serological differential diagnosis with diagnostic test systems in the format of immunochip sensitive and specific, easy to perform, eliminates the subjectivity of the assessment, due to the automated interpretation of results.

Analysis using the diagnostic test systems in the format of immunochip economically, as it replaces at least four or five diagnostic test systems other formats. In the analysis using the proposed diagnostic test systems in the format of immunochip requires a minimum amount of the material under investigation.

1. Diagnostic test-system in the format immunochip for serological differential diagnosis of syphilis, comprising immunosorbent assay with immobilized thereon separately antigens of Treponema pallidum Tr, Tr, Tmra, Tr, conjugate and reagents necessary to detect the Oia complex antigen-antibody characterized in that immunosorbent optionally immobilized cardiolipin, and the antigens of Treponema pallidum and cardiolipin immobilized in at least two repeats, and the conjugate consists of a mixture of antivitamin antibodies to human IgG and IgM antibodies to human modified in two different spectral characteristics of the fluorophores.

2. Diagnostic test system according to claim 1, wherein the immunosorbent and additionally separately immobilized at least one antigen and/or peptide Treponema pallidum selected from CR, Tr, Tr, Tr, Tr, Tr 0453 at least two repetitions.

3. Diagnostic test system according to claim 1, wherein the cardiolipin is an oxidized derivative of cardiolipin, United with protein.

4. Diagnostic test system according to claim 1, wherein the immunosorbent optionally immobilized auxiliary controls at least two repetitions.

5. Diagnostic test system according to claim 4, characterized in that the auxiliary controls are antibodies to the immunoglobulin IgG, and/or antibodies to immunoglobulin IgM, and/or immunoglobulin IgG, and/or immunoglobulin IgM, and/or internal negative control, and/or markers of the boundaries of Arreau.

6. Diagnostic test system according to claim 1, characterized in that Thu is immunosorbent is a slide in the form of a glass or plastic substrate with surface modified or coated with nitrocellulose.

7. Diagnostic test system according to claim 6, characterized in that immunosorbent made in the form of a slide into array, each of which is designed to analyze a single sample.

8. Diagnostic test system according to claim 1, characterized in that immunosorbent is a polystyrene tablet, each hole which is designed for analysis of one sample.

9. Diagnostic test system according to claim 1, characterized in that the quality of the reagents necessary for the detection of complex antigen-antibody diagnostic test system contains a diluent specimens and controls, diluent conjugate, the rinse solution immunosorbent assay, a positive control sample (K+), a negative control sample ( -).

10. The way serological differential diagnosis of syphilis with diagnostic test systems in the format of immunochip according to claim 1, comprising depositing on immunosorbent solution for cultivation specimens and controls, making the control and the samples, followed by incubation at a temperature of 20-42°C for 15-60 min for the formation of complex antigen-antibody, immunosorbent washed, next to immunosorbent contribute conjugate solution and carry out the incubation at a temperature of 20-42°C for 15-60 min, immunos is bent washed, dried, followed by detection of the formed complexes antigen-antibody and identify reginavia and treponemataceae antibodies of class G (IgG) and/or M (IgM), based on estimates of signal intensity of fluorescence upon activation of the respective channels of the multichannel fluorescence scanner.

11. The way serological differential diagnosis of syphilis according to claim 10, characterized in that immunosorbent additionally separately immobilized at least one antigen and/or peptide Treponema pallidum selected from CR, Tr, Tr, Tr, Tr, Tr 0453 at least two repetitions.

12. The way serological differential diagnosis of syphilis of claim 10, wherein the cardiolipin is an oxidized derivative of cardiolipin, United with protein.

13. The way serological differential diagnosis of syphilis according to claim 10, characterized in that the control samples represent a positive control sample (+) and negative control sample ( -).

14. The way serological differential diagnosis of syphilis of claim 10, wherein the immunosorbent and additionally immobilizer auxiliary controls at least two repetitions.

15. The way serological differential diagnosis of syphilis through 14, characterized in that the auxiliary is ontrol represent antibodies to immunoglobulin IgG person, and/or antibodies to immunoglobulin IgM, and/or immunoglobulin IgG, and/or immunoglobulin IgM, and/or internal negative control, and/or markers of the boundaries of Arreau.

16. The way serological differential diagnosis of syphilis of claim 10, wherein immunosorbent is a slide in the form of a glass or plastic substrate with surface modified or coated with nitrocellulose.

17. The way serological differential diagnosis of syphilis according to item 16, wherein immunosorbent made in the form of a slide into array, each of which is designed to analyze a single sample.

18. The way serological differential diagnosis of syphilis of claim 10, wherein immunosorbent is a polystyrene tablet, each hole which is designed for analysis of one sample.

19. The way serological differential diagnosis of syphilis according to claim 10, characterized in that the detection ragenovich antibodies is carried out in the location of cardiolipin on the immunosorbent.

20. The way serological differential diagnosis of syphilis according to claim 10, characterized in that the detection treponemataceae antibodies is carried out in places of localization of antigens of Treponema pallidum on the immunosorbent.



 

Same patents:

FIELD: biotechnology, immunology, in particular production of serum panels to control quality of test systems for lues serotological diagnosis.

SUBSTANCE: claimed panel contains set of samples with standardized content of IgG class antibodies to T. palladium p17 and p41 antigens: namely sample with prevalent content of antibodies to p17 antigen, samples with prevalent content of antibodies to p41 antigen, samples containing antibodies to p17 antigen only, samples containing antibodies to p41 antigen only, and samples containing mixture antibodies to h17 and p41 in equal concentrations. Claimed method includes sampling of positive serums based on ratio (K) of reverse titers of antibodies to T. palladium antigens with mol.w. of 17 and 41 kD (K = T-1p17/T-1p41) and sampling of original serum samples with K>=2, K<=1|2 and K = 1. Said samples are diluted with non-immune donated serum to produce necessary concentration of specific antibodies.

EFFECT: improved method for examination of test system specificity, sensitivity, and other characteristics.

7 cl, 2 tbl, 6 ex

FIELD: medicine, dermatology.

SUBSTANCE: before carrying out separate IEA for detecting anti-Treponema antibodies one should additionally treat patient's blood serum with a magnetic sorbent with an immobilized protein A of staphylococcus that sorbs IgG1 and IgG4. The innovation provides higher specificity and simplicity of the method conducted.

EFFECT: higher accuracy of diagnostics.

3 ex, 1 tbl

FIELD: medicine, infectious diseases.

SUBSTANCE: invention relates to antigenic composition and a method for detection of antibodies raised to Treponema pallidum in syphilis diagnosis. The antigenic composition comprises synthetic cardiolipin and synthetic lecithin (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and can comprise additionally cholesterol and alcohol. The antigenic composition can be used as an immunoreagent in immune analysis for detection of antibodies associated with the T. pallidum infection. The proposed method shows the enhanced sensitivity and specificity with respect to the T. pallidum infection.

EFFECT: improved detecting method.

23 cl, 8 tbl, 7 ex

The invention relates to medicine, namely to dermatology, and in particular to methods of diagnosing whether a person mycoplasmas in men

The invention relates to medicine, namely to medical Microbiology, and can be used for serological diagnosis of syphilis
The invention relates to medicine, namely, venereology, and can be used for in vitro diagnosis of false-positive reactions for syphilis due to antiphospholipid syndrome
The invention relates to medicine, namely to venereology
The invention relates to medicine, namely to microbiological research methods
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The invention relates to medicine, in particular to Microbiology, and in particular to methods of predicting the course of infectious-inflammatory diseases of the urogenital tract

FIELD: chemistry; biochemistry.

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EFFECT: use of the disclosed method provides high reliability and information content of analysis.

9 cl, 6 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: after antigen-antibody complex is prepared in a reaction compartment wherein said antigen to be modified and antibody are bound, said reaction compartment is washed with using a sample solution. The invention allows detecting a signal reflecting quantity of said antigen to be modified at precision comparable when using a washing solution to this without using said solution. Herewith, the washing solution is not required to be supplied from the outside of a chip or to be ensured therein beforehand.

EFFECT: immunoassay is easily realised on the chip.

6 cl, 6 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention can be used for evaluating the antibody level in blood serum by enzyme-linked immunosorbent assay (ELISA) in diagnostics of the diseases caused by capsular forms of such agents, as Meningococcus, Pneumococcus, Streptococcus, Haemophilus, Neisseria, Salmonella, Klebsiella, Pseudomonas, and also for evaluating the postvaccinal immunity level.

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1 dwg, 1 tbl, 3 ex

FIELD: instrument making.

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EFFECT: higher efficiency, accuracy and reliability.

162 cl, 27 dwg

FIELD: veterinary.

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EFFECT: application of claimed test-system IEA will allow to carry out simultaneously epizootological monitoring of five important infections of livestock, retrospective diagnostics of respiratory infections, and estimation of immunity stress in animals resulting from application of vaccines, determination of level of colostral antibodies in young animals in the first weeks or days of life, estimation of therapeutic medicine quality.

10 tbl

FIELD: medicine.

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3 cl, 2 dwg, 5 ex

FIELD: medicine.

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EFFECT: improved effectiveness and reduced consumption of the agent as compared to a prototype, owing to higher specific activity and effective multiple regenerability, simplified manipulations with the agent and maintained suspension of granules thereof in sorption process ensured by constant magnetic field that improves the active sorption area, and reduced destruction of perfused blood corpuscles.

5 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention refers to the biochemistry and molecular biology, in particular to the immunochemical analysis. The claimed microchip for multiple parallel immunological analysis of the compounds represents the array of three-dimensional hydrogel elements with specified volume formed on the support by the photo- or chemically induced polymerisation and containing the biological molecules (ligands) of identical or different nature. The method of quantitive immunoassay implies microchip incubation to the reaction media including the analysed sample for the purpose of complex generation. The biological microchips for quantitive immunological analysis and method for implementation of multiple parallel quantitive analysis of the compounds can be used in the analysis of the wide range of low- and high-molecular compounds, in medicine, pharmacology, food industry, environmental protection, scientific researches including proteomics.

EFFECT: possibility of multiple parallel immunological analysis of the compounds.

33 cl, 12 ex, 2 tbl, 11 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, in particular to medical diagnostics. A nano-biochip for registration of proteins and albuminous complexes and a way of its reception is offered. As a biochip substrate a non-stratified material, for example polycarbonate or polystyrene with a relief grid, is used and then the surface is leveled by means of a die providing equal surface with the size of roughnesses not more than 1 mm; after that updating and immobilisation of the molecules-probes capable to cooperate with investigated proteins and albuminous complexes on a surface of the chip specifically is carried out.

EFFECT: way allows to raise reliability of the received biochip at the expense of exception of its stratification and to raise reliability of diagnostics at the expense of rising of registration sensitivity of specific proteins and albuminous complexes.

12 cl, 2 dwg, 1 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology can be used for development of specific diagnostic aids. According to the invention the method covers producing antigen erythrocytic colibacillosis diagnosticum by extracting an adhesive antigen from Escherichia cultures, centrifuging the extract, and separating the supernatant. Thereafter formalinised tanninised animal erythrocytes are sensitised with the produced antigen. Extraction is performed in culture fluid containing Escherichia cell culture with phosphate-carbamide buffer 1.8-2.0 M of pH 7.2-7.4 at temperature 40-45°C during 25-30 min. The culture fluid containing Escherichia cell culture and phosphate-carbamide buffer are taken in mass ratio 1:0.4-0.6 respectively. After extraction the supernatant is heated up at 65-68°C within 25-30 min.

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3 ex

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11 cl, 5 dwg, 4 ex, 1 tbl

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