Tag Archives: sensitivity

Development and characterization of highly informative ELISA for the detection of IgG and IgA antibodies to Сhlamydia trachomatis

O. Yu. Galkin1, Yu. V. Gorshunov1, O. B. Besarab1, O. M. Ivanova2

1National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”;
e-mail: alexfbt@gmail.com;
2Xema Ltd., Kyiv

The goal of this work was developing of highly informative an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgA antibodies against to Chlamydia trachomatis, as well as comparative characterization of developed assay using standardized control materials. The study was conducted using: monoclonal antibodies (McAbs) to human IgA and IgG; recombinant Ch. trachomatis proteins – Pgp3, major outer membrane protein (MOMP); two panels of characterized sera and four reference ELISA kits. The study of immunochemical activity of peroxidase conjugates of McAbs was performed in comparison with conjugates of commercial analogues: anti-IgG McAb 2A11 and anti-IgA McAb AD3. About half of the conjugates from the received McAbs panel were more active compared to the reference antibody conjugates. It was quite justified to use the conjugates of antibodies that interact with different antigenic determinants. When IgG antibodies were detected to MOMP, it was justified 1.14-1.56 times more; when IgA antibodies were detected to MOMP, it was justified 1.16-1.37 times more. ELISA for detecting IgG/IgA antibodies to MOMP and Pgp3 of Ch. trachomatis were evaluated using appropriately described serum panels OCO-42-28-313-00 and OCO-42-28-314-00. Comparative studies of the developed ELISA for the detection of IgG and IgA antibodies to the MOMP and Pgp3 of Ch. trachomatis showed their prominent advantage over the commercial analogues, which more clearly demonstrates the difference in the ratio of average values of optical density of positive and negative samples of the described panel of sera: this indicator for commercial kits was 1.36-3.59 times less.

Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60)

O. Yu. Galkin, A. B. Besarab, T. N. Lutsenko

National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”;
e-mail: alexbio@mail.ua

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 negative, 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.