RU2394496C1 - Syphilis diagnostic technique by simultaneous detection of reaginic and treponema-specific t pallidum antibodies on microscope aldehyde slides - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention refers to medicine, particularly to infectious diseases. It involves simultaneous analysis of blood serum for reaginic and treponema-specific antibodies to cardiolipin antigen and recombinant T pallidum protein complex with molecular weight 15, 17, 39, 41, 42, 44.5, 47 kDa, immobilised on microscope aldehyde slides to detect antibodies by a conjugate solution containing Cy5 phosphor tagged human IgG antibodies and Cy3 phosphor tagged human IgM antibodies; slide scanning in a multichannel biochip scanner; automatic data analysis in a program used to convert fluorescent signals to digital positiveness coefficients; and qualitative presentation. Reaginic and treponema-specific antibodies found in a sample indicate syphilis, while no reaginic and treponema-specific antibodies found in the sample shows the absence of syphilis in a patient. ^ EFFECT: method allows for differentiated detection of antibodies to the most immunogenic T Pallidum antigens produced at the different stages of disease, for the objective automated data record, for cutting examination and diagnosing time. ^ 4 cl, 2 ex

Description

The technical field of the present invention

The present invention relates to medicine, to the diagnosis of sexually transmitted infections, namely syphilis.

BACKGROUND OF THE INVENTION

Syphilis is an infectious disease caused by pale treponema (T. pallidum), transmitted mainly through sexual contact, characterized by damage to the skin, mucous membranes, nervous system, internal organs and musculoskeletal system, often leading to disability and even death (in case of development late forms of syphilitic infection - cardiosyphilis, neurosyphilis; with the transmission of syphilis from mother to fetus and the development of congenital syphilis). Syphilitic infection is included in the list of socially significant diseases and diseases that pose a danger to others [1].

The high incidence of syphilis in the Russian Federation [2] determines the relevance of developing new methods for diagnosing this infection.

It is known that in the diagnosis of syphilitic infection, serological research methods play the leading role, the principle of which is to determine the immune antibodies associated with syphilitic infection in the blood serum (plasma, cerebrospinal fluid) of patients. The diagnosis of syphilis is complex, which is due to the variability of the macroorganism’s immune response at different stages of syphilitic infection and is expressed by a change in the antibody content with respect to individual antigenic structures (determinants) of the syphilis pathogen, which can be detected using different research methods.

Depending on the type of antibodies detected and the antigen used, serological reactions for the diagnosis of syphilis are usually divided into non-treponemal (NTT) and treponemal (TT). Nontreponemal tests (microprecipitation reaction - RMP, rapid plasma reactin test - RPR) determine reagin antibodies to the cardiolipin antigen and are used mainly for screening for syphilitic infection and in assessing the effectiveness of specific treatment. Treponemal tests (passive hemagglutination reaction - RPHA, immunofluorescence reaction - RIF, immobilization reaction of pale treponemas - RIBT, enzyme-linked immunosorbent assay - ELISA) reveal treponemospecific antibodies and are confirmatory [3].

NTTs are non-standardized methods for the diagnosis of syphilis, a significant role in the interpretation of their results is played by the subjective component. In addition, when performing the most commonly used RMP, each laboratory prepares the cardiolipin antigen on its own; while the quality of the antigen may depend on the correct preparation, conditions and the duration of its storage. Diagnostics for stating RMP differ both in quality and in quantitative terms, which undoubtedly affects the result of the reaction [3].

Also, NTTs most often give "false positive" [4-9] and "false negative" [10-13] test results for syphilis.

The cardiolipin antigen used in nontreponemal tests is a mixture of highly purified lipid substances of animal origin; its composition and the ratio of its constituent components are quite well known (cardiolipin 0.03%, lecithin 0.27%, cholesterol 0.9%) [14].

A treponemal test is required when obtaining positive NTT results [3]. At the same time, all TTs in some cases can give “false positive” and “false negative” results and exhibit different sensitivity at different stages of infection [4, 15-21]. In addition, the RIF and RIBT, which are currently arbiter reactions for the diagnosis of syphilis, are difficult to standardize, a subjective factor has a significant impact on the results of the study, their formulation requires the maintenance of vivarium and inoculation of a live culture of pathogenic microorganisms (T. pallidum), which is time consuming and epidemiologically dangerous. RPGA is a standardized reaction: all components are produced as a set of industrial reagents, however, the test results are read visually, which can cause errors in interpreting the reaction results.

The most optimal technology for the diagnosis of syphilis is currently considered to be an ELISA carried out by a set of reagents for industrial production with automation of a number of processes: washing procedures, recording and evaluation of the results.

All of the above technologies are based on the identification of a total pool of antitreponemal antibodies to T. pallidum antigens in a sample of biological material of a patient with different molecular weights varying from 15 to 97 kDa, differing in their immunogenicity, that is, the ability to detect an immune response to the introduction of pale treponema into the body at different stages of syphilitic infection.

In recent years, immunoblotting methods based on the ELISA principle and aimed at detecting antibodies to each pale treponema antigen differentially have appeared, which significantly increases the sensitivity and specificity of the method [22-24]. It is possible to evaluate the contribution of each antigen to the formation of the total immune response, to evaluate the effectiveness of specific treatment of various forms of syphilis [25]. The method of immunoblotting practically does not give false positive and cross reactions with other treponemas [22, 26-33].

Many test systems based on the principle of enzyme-linked immunosorbent assay are not very sensitive and specific, and can detect antibodies to the causative agent of syphilis only at some specific stage of infection. In primary syphilis, the earliest response to the introduction of T. pallidum into the human body is determined by TpN47 and somewhat later by TpN15 and TpN17 [34]. Zugehor M. et al. (1989) found pronounced reactogenicity of the 39 kDa polypeptide with the sera of patients with primary and secondary syphilis [35]. George R et al. (1998) recommended the use of immunoblotting using antigens 47, 17 and 15.5 kDa for the diagnosis of primary and secondary syphilis. They noted a lower sensitivity of the method in patients with latent syphilis [36]. According to domestic and foreign authors, antibodies to TpN15, TpN17, TpN44.5 (TmpA) and TpN47 are most significant in the diagnosis of syphilis [21, 36-40].

Thus, the data presented indicate that so far the most known immunogenic recombinant T. pallidum antigens that can record the immune response at different stages of syphilitic infection are antigens with a molecular weight of 15, 17, 39, TmpA (41 kDa, 42 kDa, 44.5 kDa) and 47 kDa.

The method of immunoblotting can be a convenient tool for early diagnosis of syphilis, differential diagnosis of various stages of the disease, and also serve as the basis for the development of new tests for syphilis by choosing priorities for the use of certain T. pallidum antigens at different stages of syphilitic infection.

The classical algorithm for examining a patient for syphilis using serological tests consists of staging a nontreponemal test and, with a positive result, one or two treponemal tests. However, NTT and TT are produced in a different format and are performed for different times after receiving and preparing for the study of blood serum: RMP - within 10-15 minutes; RPGA - from 40 minutes to 2 hours; ELISA 3-4 hours, RIF, RIBT and immunoblotting - at least 18 hours [41, 42]. Non-treponemal and treponemal tests are often performed on different days and even in different laboratories. In addition, due to the influence of the subjective component in determining the results of both NTT and TT, a dermatovenerologist often encounters a situation where the interpretation of the results of serological reactions is difficult, which requires repeated or additional examination. As a result, the period from the initial treatment of the patient to the establishment of the final diagnosis can be up to 2 weeks or more, which is extremely undesirable in relation to an epidemiologically dangerous, socially significant disease.

This situation could have been avoided by developing a standardized method for the diagnosis of syphilis, which allows for the simultaneous determination of reagin and treponemal antibodies within a single research format, for one period of time.

In recent years, publications have appeared about the use for the diagnosis of various diseases, including infectious, protein microarrays or immunochips [43-51]. The use of protein microarrays significantly reduces the time and financial costs spent on research. The method allows the study of biological material obtained from several patients simultaneously. An immunochip is a carrier plate (glass, polyacrylamide gel, nylon membrane), on which cells with fixed samples of antigens are located in a certain order.

For syphilitic infections, recombinant pale treponema proteins can be used as antigens. There are few publications on the development of an experimental test system in the immunochip format for serological diagnosis of syphilis, not inferior in sensitivity and specificity of ELISA, RPHA and immunoblotting, as well as to address the need for additional treatment [52, 53]. For immobilization on slides, 4 recombinant T.pallidum proteins were obtained (Tp 47, Tp 17, Tp 15, TmpA). Each antigen corresponded to an individual spot (spot). The principle of the test system is based on an indirect method for detecting specific antibodies to the pathogen using fluorescence detection. On one erray (analogue of a plate hole, strip), it is possible to simultaneously detect both IgG and IgM antibodies using a mixture of human antispecific conjugates modified with TRITC and FITC fluorophores, respectively. To analyze the specificity of the test system, sera from patients with Lyme borreliosis and leptospirosis were used. The developed test system showed 100% sensitivity and 100% specificity. Such an immunochip can increase the efficiency of patient examination due to simultaneous screening for syphilis and detection of the spectrum of antibodies to T. pallidum due to separate immobilization of antigens on the slide [52].

At present, in the Russian Federation there are no diagnostic methods for syphilitic infection that combine the capabilities of nontreponemal and treponemal tests and allow detecting antibodies differentially to the most immunogenic T. pallidum antigens produced at different stages of the disease, simultaneously detecting reagin antibodies, and conducting objective automated recording of results , reduce the time of examination of the patient and the timing of the final diagnosis.

Disclosure of the present invention

The essence of the present invention is a method by which in a single research format for the same time in a short time it is possible to detect syphilis at any stage and in any form of the course of infection, which allows objective automated recording of research results, eliminating the influence of the subjective factor.

Thus, the present invention relates to a method for the diagnosis of syphilis by simultaneously determining the patient's serum reagin and treponemospecific antibodies to a cardiolipin antigen and a complex of recombinant T. pallidum antigens immobilized on an immunosorbent, comprising the following stages:

a) obtaining test samples of blood serum;

b) incubation of the immunosorbent with the added samples of the test blood serum obtained in stage a), and a conjugate containing a fluorophore-labeled antibody to human IgG and another fluorophore-labeled antibody to human IgM,

c) analysis of the obtained fluorescent signals for the presence or absence of reagin and treponemospecific antibodies to T. pallidum in the sample, the presence of reagin and treponemospecific antibodies in the sample indicates the presence of syphilis, and the absence of reactin and treponemospecific antibodies in the sample indicates the absence of syphilis in the patient.

In accordance with one of the embodiments of the present invention, the test samples of blood serum receive a standard method for conducting serological studies.

In accordance with one preferred embodiments of the present invention, the immunosorbent is microscopic aldehyde slides in the format of an immunochip with T. pallidum antigens immobilized on them with a molecular weight of 15, 17, 39, 41, 42, 44.5, 47 kDa and cardiolipin, and also three internal controls on the validity of the system. The immunosorbent is incubated with added test serum samples, positive and negative controls, and then with a working solution of a conjugate containing antibodies to human IgG labeled with fluorophore Cy5 and antibodies to human IgM labeled with fluorophore Cy3, respectively, added to the samples and controls. Slides are washed with a washing solution (at the stage of preparation of the immunosorbent, after incubation of blood serum and conjugate samples with the immunosorbent), dried and scanned on a multi-channel biochip scanner, while fluorescence of samples containing antibodies to T. pallidum is noted.

The results are processed automatically using a program that translates fluorescent signals into digital indicators of the coefficient of positivity. In conclusion, a qualitative research result is obtained: positive in the case of the presence of reagin and treponemospecific antibodies to T. pallidum in the sample, negative in the absence of reactin and treponemospecific antibodies to T. pallidum in the sample.

Embodiments of the present invention

Example No. 1. Patient A. with a diagnosis of “Syphilis secondary of the skin and mucous membranes” (the diagnosis was confirmed by the results of clinical and laboratory studies - the positive results of dark-field microscopy in the study of syphilitic rashes on the skin and the positive results of serum serum reactions - RMP, RPGA, ELISA) were the standard way blood serum obtained; an immunosorbent was prepared, which is microscopic aldehyde slides with immobilized T. pallidum antigens with a molecular weight of 15, 17, 39, 41, 42, 44.5, 47 kDa and cardiolipin, as well as three internal controls for the validity of the system; washing of the immunosorbent with a working solution for washing was carried out by adding 100 μl of a washing solution to each well of immunosorbent, followed by incubation on a thermo-shaker with a rotating platform (500 rpm) at 37 ° C for 2-3 minutes; excess washing solution was removed by carefully aspirating the contents from the wells using a biochip mower or a medical vacuum aspirator; drying the immunosorbent at room temperature; in two wells of the immunosorbent (the reaction was set in duplicate), 90 μl of the dilution solution were added, as well as 10 μl of the tested blood serum that was not subjected to inactivation by incubation at elevated temperature (+ 56 ° C); 10 μl of the positive control (K +), which is the blood serum of a patient with syphilis, containing antibodies to the pathogen) was introduced into one of the wells of the immunosorbent); 10 μl of the negative control (K-), which is a blood serum that does not contain antibodies to the causative agent of syphilis, was added to another well of the immunosorbent; the immunosorbent with a closed lid was incubated for 30 min on a thermal shaker with a rotating platform (500 rpm) at 37 ° C; washing of the immunosorbent was carried out by adding 100 μl of the working solution for washing and incubation on a thermal shaker with a rotating platform (500 rpm) at 37 ° C for 2-3 minutes; then, 60 μl of a pre-prepared working solution of the conjugate containing antibodies to human IgG labeled with Cy5 fluorophore and antibodies to human IgM labeled with Cy3 fluorophore, respectively, were added to the wells; immunosorbent with conjugate was incubated for 30 min on a thermo-shaker with a rotating platform (500 rpm) at 37 ° С; excess washing solution was removed by carefully aspirating the contents from the wells using a biochip mower or a medical vacuum aspirator; washing of the immunosorbent with a working solution for washing was carried out by adding 100 μl of a washing solution to each well of immunosorbent, followed by incubation on a thermo-shaker with a rotating platform (500 rpm) at 37 ° C for 2-3 minutes; the immunosorbent was rinsed with distilled water and the slides were dried by centrifugation for 1 min at 1000 rpm. After that, the immunosorbent was scanned on a multi-channel scanner for biochips by activating the channels Su5 and Su3; scanned fluorescence images are saved in tif format and opened in a special program with software for image interpretation; using the program, the fluorescent signals of patient A. samples were converted into digital indicators of the positivity coefficient; the patient received a positive test result for the presence of treponemospecific IgG and IgM to the causative agent of syphilis, as well as antibodies to cardiolipin, which indicated the presence of antibodies to T. pallidum in the sample.

Thus, the use of this method allowed patient A. to confirm syphilis.

Example No. 2. In patient V., during the examination of which the diagnosis of Syphilis secondary of the skin and mucous membranes was not confirmed (a negative result of dark-field microscopy in the study of skin rashes and a negative result of serum blood serum reactions - RMP, RPGA, ELISA), serum was obtained in a standard way blood an immunosorbent was prepared, which is microscopic aldehyde slides with immobilized T. pallidum antigens with a molecular weight of 15, 17, 39, 41, 42, 44.5, 47 kDa and cardiolipin, as well as three internal controls for the validity of the system; washing of the immunosorbent with a working solution for washing was carried out by adding 100 μl of a washing solution to each well of immunosorbent, followed by incubation on a thermo-shaker with a rotating platform (500 rpm) at 37 ° C for 2-3 minutes; excess washing solution was removed by carefully aspirating the contents from the wells using a biochip mower or a medical vacuum aspirator; drying the immunosorbent at room temperature; in two wells of the immunosorbent (the reaction was set in duplicate), 90 μl of the dilution solution were added, as well as 10 μl of the tested blood serum that was not subjected to inactivation by incubation at elevated temperature (+ 56 ° C); 10 μl of the positive control (K +), which is the blood serum of a patient with syphilis, containing antibodies to the pathogen) was introduced into one of the wells of the immunosorbent); 10 μl of the negative control (K-), which is a blood serum that does not contain antibodies to the causative agent of syphilis, was added to another well of the immunosorbent; the immunosorbent with a closed lid was incubated for 30 min on a thermal shaker with a rotating platform (500 rpm) at 37 ° C; washing of the immunosorbent was carried out by adding 100 μl of the working solution for washing and incubation on a thermal shaker with a rotating platform (500 rpm) at 37 ° C for 2-3 minutes; then, 60 μl of a pre-prepared working solution of the conjugate containing antibodies to human IgG labeled with Cy5 fluorophore and antibodies to human IgM labeled with Cy3 fluorophore, respectively, were added to the wells; immunosorbent with conjugate was incubated for 30 min on a thermo-shaker with a rotating platform (500 rpm) at 37 ° С; excess washing solution was removed by carefully aspirating the contents from the wells using a biochip mower or a medical vacuum aspirator; washing of the immunosorbent with a working solution for washing was carried out by adding 100 μl of a washing solution to each well of immunosorbent, followed by incubation on a thermo-shaker with a rotating platform (500 rpm) at 37 ° C for 2-3 minutes; the immunosorbent was rinsed with distilled water and the slides were dried by centrifugation for 1 min at 1000 rpm. After that, the immunosorbent was scanned on a multi-channel scanner for biochips by activating the channels Su5 and Su3; scanned fluorescence images are saved in tif format and opened in a special program with software for image interpretation; using the program, the fluorescent signals of patient B.'s samples were converted into digital indicators of the positivity coefficient; the patient was given a negative test result for the presence of treponemospecific IgG and IgM to the causative agent of syphilis, as well as antibodies to cardiolipin, which indicated the absence of antibodies to T. pallidum in the sample.

Thus, the use of this method allowed patient B. to exclude syphilis.

Information sources

Claims (4)

1. A method for the diagnosis of syphilis by simultaneously determining the patient's serum reagin and treponemospecific antibodies to the cardiolipin antigen and the complex of recombinant T. pallidum antigens immobilized on an immunosorbent, comprising the following stages:
a) obtaining test samples of blood serum;
b) incubation of the immunosorbent with the added samples of the test blood serum obtained in stage a), and a conjugate containing a fluorophore-labeled antibody to human IgG and another fluorophore-labeled antibody to human IgM,
c) analysis of the obtained fluorescent signals for the presence or absence of reagin and treponemospecific antibodies to T. pallidum in the sample, the presence of reagin and treponemospecific antibodies in the sample indicates the presence of syphilis, and the absence of reactin and treponemospecific antibodies in the sample indicates the absence of syphilis in the patient. 2. The method according to claim 1, according to which blood serum samples are obtained in a standard manner for conducting serological studies. 3. The method according to claim 1, wherein the immunosorbent is microscopic aldehyde slides in the format of an immunochip with T. pallidum antigens immobilized on them with a molecular weight of 15, 17, 39, 41, 42, 44.5, 47 kDa and cardiolipin , as well as three internal controls on the validity of the system. 4. The method according to claim 1, whereby antibodies to human IgG and antibodies to human IgM are labeled with a Cy5 fluorophore and Cy3 fluorophore, respectively.