CharaCteristiCs of enzyme-linked immunosorbent assay for deteCtion of igg antibodies speCifiC to Сhlamydia trachomatis heat shoCk protein (hsp-60)

The goal of this work was to study sensitivity and specificity of the developed ELISA set for the identification of IgG antibodies against Chlamydia trachomatis HSP-60 (using biotinylated tyramine-based signal amplification system). The study was conducted using a panel of characterized sera, as well as two reference elISa sets of similar purpose. according to the results of elISa informative value parameters, the elISa we have developed showed the highest specificity and sensitivity parameters (no false negative or false positive results were registered). In 4 out of 15 intralaboratory panel serum samples initially identified as nega tive, anti-HSP-60 IgG-antibodies test result in reference ELISA sets upon dilution changed from negative to positive. The nature of titration curves of false negative sera and commercial monoclonal antibodies А57-В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of anti-idiotypic antibodies in these samples. Upon sera dilution, idiotypic-anti-idiotypic complexes dissociated, which caused the change of test result. High informative value of the developed ELISA set for identification of IgG antibodies against C. trachomatis HSP-60 has been proven. Anti-idiotypic antibodies possessing C. trachomatis anti-HSP-60 activity and being one of the causes of false negative results of the relevant elISa-based tests have been identified in blood sera of individuals infected with chlamydial genitourinary infection agents.


e x p e r i m e n t a l w o r k s e x p e r i m e n t a l w o r k s
UDC 573.6.083.3+573.6 The goal of this work was to study sensitivity and specificity of the developed ELISA set for the identification of IgG antibodies against Chlamydia trachomatis HSP-60 (using biotinylated tyramine-based signal amplification system).The study was conducted using a panel of characterized sera, as well as two reference elISa sets of similar purpose.according to the results of elISa informative value parameters, the elISa we have developed showed the highest specificity and sensitivity parameters (no false negative or false positive results were registered).In 4 out of 15 intralaboratory panel serum samples initially identified as nega tive, anti-HSP-60 IgG-antibodies test result in reference ELISA sets upon dilution changed from negative to positive.The nature of titration curves of false negative sera and commercial monoclonal antibodies А57-В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of anti-idiotypic antibodies in these samples.Upon sera dilution, idiotypic-anti-idiotypic complexes dissociated, which caused the change of test result.High informative value of the developed ELISA set for identification of IgG antibodies against C. trachomatis HSP-60 has been proven.Anti-idiotypic antibodies possessing C. trachomatis anti-HSP-60 activity and being one of the causes of false negative results of the relevant elISa-based tests have been identified in blood sera of individuals infected with chlamydial genitourinary infection agents.K e y w o r d s: enzyme-linked immunosorbent assay, Chlamydia trachomatis, heat shock protein, anti-idiotypic antibodies, sensitivity, specificity.
C hlamydial genitourinary infection (GUI) is one of the most common sexually transmitted infections.According to World Health Organization, Chlamydia trachomatis infects about 90 million people each year by sexual transmission.In Ukraine, the rate of chlamydial GUI is 80 cases per 100,000 population.Nearly 16% of pregnant women are infected with C. trachomatis.About 50-60% of tubular infertility cases are caused by chlamydial infection.A quarter of all ophthalmic and respiratory diseases in newborn and younger children are associated with chlamydial infection [1,2].
Effective diagnosis is one of important components of chlamydial GUI control, which can be realized through both direct (detection of antigens, nucleic acids, pathogenic agent microscopic examination and cultivation) and indirect (specific antibo dies detection) techniques.One of the commonly used methods in diagnostics is enzyme-linked immunosorbent assay (ELISA) that allows differential diagnosis -determination of disease stage and course, which is especially important in chronic conditions.For that purpose blood serum (plasma) and human biological fluids are tested for the presence of IgM, IgA and IgG classes of specific antibodies to pathogen's antigens [2,3].
Prolonged persistency of C. trachomatis increases the expression of heat shock protein with molecular mass 60 kDa (HSP-60), which is highly homologous with respective natural protein in human cells.Its production results in molecular mimicry phenomena, which in turn triggers autoimmune processes.Increased level of immune system stimulation by heat shock protein that occurs in case of reinfection or persistent infection leads to chronic inflammation and scarring of tissues and may play a role in pathogenesis of endometrial and uterine adnexal lesions.These immunopathological reactions may be the cause of ectopic pregnancy and tubular infertility [4,5].Thus, examination of patients infected with C. trachomatis for the presence of IgG antibodies to HSP-60 is essential.
The relevance of the work to improve the sensitivity of ELISA for detection of IgG antibodies to HSP-60 is linked to the fact that HSP-60 is a marker of autoimmune processes (complications of urogenital infections).Such tests are carried out after primary diagnosis, including the use of molecular genetic techniques (e.g.polymerase chain reaction, PCR).
Earlier in the study, we obtained biologic components for development of ELISA set in order to carry out qualitative (semiquantitative) detection of IgG antibodies to HSP-60 C. trachomatis [4,6].
This study aimed at evaluation of diagnostic characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies to HSP-60 С. trachomatis.

materials and methods
Blood sera.Blood sera of persons infected with C. trachomatis (dia gnosis confirmed by PCR) were used in the study.
Indirect enzyme-linked immunosorbent assay.Recombinant HSP-60 C. trachomatis adsorption was performed in 0.05 M carbonate-bicarbonate buffer solution (pH 9.6) for 12 h at 4 °С at concentration of 4 μg/ml.Free areas on surface of plates' wells were filled by bovine serum albumin solution.
The wells were injected with 100 μl of studied human blood sera and incubated at 37 °С for 1 h, then washed out three times with 0.02 M phosphatebuffered saline with Tween20, pH 7.27.4(PBST).To detect specific antigens, there was used conjugate of monoclonal antibodies to human IgG with horseradish peroxidase incubated at 37 °С for 30 min.After incubation the plate was washed out with PBST and injected with chromogenic substrate solution (3,3′,5,5′tetramethylbenzidine, 0.003% Н 2 О 2 , 0.15 М citrate bufferred solution, рН 5.0).Following the enzymatic reaction stop, optical density (ОD) of solution was measured at wavelength 450 nm.
Indirect enzyme-linked immunosorbent assay with biotinylated tyramine reagent was carried out using the same procedure, however after sera incubation the wells were first injected with biotinyla ted tyramine reagent ELAST ELISA Amplification System (PerkinElmer, USA) according to instruction manual.

results and discussion
Due to their absence in the market of control materials, it was impossible to use standardized serum sets for the evaluation of ELISA diagnostic characteristics during detection of antibodies to HSP-60 C. trachomatis.In this case, the only availab le methodological approach is formation of our own intralaboratory (or in-process) serum sets and performance of comparative analysis using similar purpose diagnostic ELISA kits manufactured by various companies.
To produce our own serum set negative to specific antibodies to C. trachomatis, obtained sera were tested for the presence of specific IgG antibo dies to pathogenic HSP-60 using ELISA kits #1 and #2.The pool of negative sera inclu ded those exhibi ting positive results during testing for antibodies to other antigens of pathogen (major outer membrane protein, Pgp3).This technique allowed formation of intralaboratory set consisting of 15 positive and 20 negative sera.
In order to substantiate the modification of ELISA for detection of IgGantibodies specific for HSP-60 С. trachomatis it makes sense to address the following literature data.
First, various authors reported on possible divergence of results obtained in different tests (serum, molecular genetic, culture-based tests) during diagnosis of chlamydial genitourinary infection [7][8][9][10][11].These data suggest various informative values of the said clinical laboratory examination techniques.At the same time, there is no consensus on the nature of such discrepancies.For instance, negative results of serum tests in some patients appear quite strange in the presence of positive results for detected pathogen's nucleic acids in urogenital smear by PCR.The most common explanation of this fact relates to low test sensitivity or low level of humoral immune response to pathogen.Secondly, currently available scientific data on heat shock proteins interaction with immune system make it possible to content that HSP is the element of macro organism immune regulatory network similarly to idiotypic and anti-idiotypic system.This can be proven by following facts: their availability in both eukaryotic and prokaryotic organisms; presence of highly conserved sequences in the structure of HSP, which are also immunodominant; high immunologic activity of HSP.The HSPdependent system (network) of inflammation and autoimmu nity stimulation and regulation includes, at any rate, following elements: HSP as polyreactive antigens; natural auto antibodies and anti-idiotypic antibodies; proteins of macro organism as such released from the cells due to various reasons; foreign proteins (including HSP of microorganisms); antigen-presenting cells that can be activated by HSP; effector and regulatory T-lymphocytes interacting with HSP [12][13][14][15].
Thirdly, there are numerous reports in modern scientific literature on idiotypic and antiidiotypic interactions in blood serum of mammals and humans, including rather successful, in our opinion, attempts of mathematic modelling of this interaction, similarly to ligand-receptor interaction [16][17][18].Original approach to detection of idiotypic and anti-idiotypic interactions in polyclonal sera involves synchronic changes in receptor and ligand concentrations (primary and anti-idiotypic antibodies) in the studied system (serum) [17,18].This approach allows us to determine theoretically the concentration of antigenantibody complex (сі) depending on the dilution ratio of the serum (1) , (1) where d i -serum dilution factor, l -initial ligand (antigen) concentration, r 0 -initial concentration of bivalent receptor (antibody), k -constant of antigenantibody reaction equilibrium (invariable of antibody affinity) [18].
It is also possible to calculate within this theory [18] following probabilities: 1) both centers of any receptor are bound to ligand ( f 1 ); 2) both binding centers of any bivalent receptor are free ( f 2 ); 3) when the equilibrium is reached one of the binding centers of receptor will become free while the other -bound to ligand ( f 3 ).The said probabilities are also dependent on the dilution ratio of sera di (2-4): , , Graphic presentation of free antibodies concentrations dependence in their mixture with antigen on dilution ratio of the mixture (serum) is given in Fig. 1.
Based on the above, further investigations with regard to the development of ELISA for detection of IgG class antibodies specific for HSP60 С. trachomatis were performed in two directions: firstly, development of classic indirect ELISA for detection of IgG-antibodies (our reference designation: ELISAHSPIgG); secondly, development of indirect ELISA using biotinylated tyramine reagent (our reference designation: ELISA-HSP-IgG-TB), which efficiency we have already proved [19].In the latter case, analytemediated enzyme activating system of signal amplification allows performing studies of diluted sera, which, in turn, creates opportunities for Fig. 1.Theoretical dependence of free bivalent antibodies concentrations in their mixture with antigen on the dilution ratio of such mixture d i -fold [18]  The first stage of the study included comparative characteristic of the developed classical indirect ELISA (ELISA-HSP-IgG) with ELISA-kits by various manufacturers.Obtained results (Tables 12) show that informative value of our developed ELISA is not inferior to commercial equivalents and in some ways even superior to them.
Following stage of the study comprised testing of diluted (1:4…1:64) negative sera of intralaboratory serum set in developed ELISA using biotinylated tyramine reagent (ELISA-HSP-IgG-TB).It should be noted that testing of diluted sera was carried out following their incubation in wells of ELISA plates at 4 °С for 24 h.For all the samples, except 4 which were initially identified as negative, negative results were obtained during titration of sera of intralaboratory serum set.It is worth noting that all these sera were positive with regard to IgG antibodies to МОМР/Pgp3 C. trachomatis.The testing results of falsenegative samples of intralaboratory serum set are given in Table 3. Titration curves of these sera compared to titration of monoclonal antibody A57-B9 (Thermoscientific, USA), specific for HSP60 С. trachomatis, are given in Fig. 2.
The results of the study show that sera of intralaboratory serum set No.22, 26, 27 and 31 contain certain factors able to block the anti-HSP-60 antibodies when undiluted.The titration curves compared to MABs specific for HSP60 of chlamydial genitourinary infection allows concluding that antiidiotypic antibodies are likely to be present in these sera, which constitute the factor blocking the activity of anti-HSP-60 antibodies.
These data provide an answer, at least one of possible answers, to the question concerning falsenegative results of ELISA testing on the presence of antibodies specific for C. trachomatis in patients with confirmed diagnosis by PCR.It should be noted that obtained results point to the need to use less convenient in terms of ergonomics schemes of ELISA testing for respective diagnostic kits, which includes prolonged samples incubation and their titration.This task can obviously be completed only using ELISA kits build on effective signal amplification systems.
As was mentioned earlier, existing literature data [711] are indicative of rather frequent variations in results of different laboratory tests of chlamydial genitourinary infection, in particular, molecular-genetic, serological, and microbiological ones.This situation is rather unfavorable from the viewpoint of practical medicine and requires elevation of informative value (reliability) of laboratory methods for diagnostics of this infection.False positive and false negative results of serological tests may be obtained due to not only bioanalytical characteristics (limitations) of the test method itself (related with properties of biological reagents and "structure" of ELISA itself), but also to the presence of substances in the test material which bias the test results in various ways.Thus, e.g.influence of antigen mimicry on informative value of HIV infection serological diagnostics has been described [20].
In view of several particulars of formation of B-cell response to heat shock proteins [12][13][14][15], as well as reports on potential effects of antiidiotypic serum antibodies on the nature of immunochemical reactions (e.g.socalled prozone effect in tests based on agglutination or precipitation reaction) [16][17][18]; additionally, development of indirect ELISA for identification of anti-HSP-60 IgG-antibodies using biotinylated tyramine reagent was performed.This modification allowed carrying out identification of specific antibodies in dilute serum samples (1:4÷1:64), and, respectively, measuring the content of anti-idiotypic antibodies in them.In 4 out of 15 intralaboratory panel (ILP) serum samples initially identified as negative, antiHSP60 IgGantibodies test result upon dilution changed from negative to positive.The nature of titration curves of false negative ILP sera and commercial МАB А57В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of antiidiotypic antibo dies in these samples.Upon sera dilution, idio typicanti-idiotypic complexes dissociated that caused the change of test result.
The results of these studies, at first, are indicative of the cause (at least one of the causes) of false negative results of chlamydial genitourinary infection serological tests, and, at second, are the prere quisites for the development of new strategy of complex examination of patients for the presence of C. trachomatis infection.Obviously, in case of positive PCR test result and negative serological test result of chlamydial genitourinary infection, additional testing of such samples in a series of dilutions is expedient.To solve this problem, highly sensitive ELISA sets are to be used, for example, employing biotinylated tyraminebased signal amplification system.In our opinion, the issue of clinical rele-

Fig. 2 .
Fig. 2. Titration curves of false-negative sera for intralaboratory serum set and МАТ А57-В9 to HSP-60 C. trachomatis following incubation at 4 °С for 24 h (effect on the ability of antibodies to bind to adsorbed antigen on plate)

CharaCteristiCs of enzyme-linked immunosorbent assay for deteCtion of igg antibodies speCifiC to Сhlamydia trachomatis heat shoCk protein (hsp-60)
.086.8 Note. * Specified are arithmetic mean values for OD for each sample in three repetitions; р < 0.05T a b l e 2. Comparative analysis of various ELISA for detection of IgG-antibodies to C. trachomatis HSP-60 Note.Specified are arithmetic mean values for OD for each sample in three repetitions; р < 0.05