Synthetic positive controls for ELISA test kits for detection of IgA and IgM antibodies to Chlamydia trachomatis

Abstract

The enzyme-linked immunosorbent assay (ELISA) is the most informative and versatile method of serological diagnostics. The possibility of detecting by ELISA specific antibodies of different classes allow to differentiate primary infectious process and its remission, exacerbation and chronic disease (holding of differential diagnosis). This approach is implemented in the methodology for evaluation of patients for presence of humoral immune response against the causative agent of urogenital chlamydiosis. As with other infections immediately after Chlamydia trachomatis infection the specific IgM antibodies are formed, and subsequently basic projective antibodies of IgG class are synthesized. However, at exacerbation of chronic urogenital chlamydiosis specific IgA antibodies can be synthesized. That is why comprehensive evaluation of patients for presence of humoral immune response to Ch. trachomatis involves plasma testing of specific antibodies of all three classes. The essential problem in the production of ELISA diagnostic kits is obtaining of positive control. The classic version of positive control is human blood plasma containing specific antibodies. But specific IgM- and IgA-positive sera are deficit raw materials. This fact can significantly limit the production of diagnostic kits, especially in case of large-scale manufacture. We have suggested methodological approach to use of synthetic positive controls in indirect ELISA kits based on conjugate of normal human IgM (IgA) and monoclonal antibodies against major outer membrane protein of Ch. trachomatis. It was found that it’s possible to realize such task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and reductive amination-mediated conjugation (by sodium periodate). It was found that synthetic positive controls obtained by different methods are characterized by higher titer compared to IgM- and IgA-positive high-titer serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics, at the release time and after one-week storage at37 °C.

New monoclonal antibodies to the Chlamydia trachomatis main outer membrane protein and their immunobiological properties

“…One of them is a comparative epitope speci ficity technique. We have used such approach many times [4,5,16] when different bioanalytical methods were developed (ELISA for determination of human IgE, IgM, and IgA). In these cases, absolute epitopic specificity is not important; for the develop ment of the analytical methods, it was important to understand the ratio of the specificity of various antibodies.…”

“…One of the commonly used methods in diagnostics is enzymelinked immunosorbent assay (ELISA) that allows doi: https://doi.org/10.15407/ubj91.03.090 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 [3,4].…”

“…This circumstance can significantly limit the production of diagnostic kits, especially in the context of large-scale production. In order to increase the efficiency of ELISA test kits production, we proposed a methodological approach that addresses the use of synthetic PCs: for the ELISA test kit for the diagnosis of urogenital chlamydia, based on the principle of indirect ELISA,the use of a conjugate of IgM (IgA) normal immunoglobulins and monoclonal antibodies (McAbs) to C. trachomatis major outer membrane protein (MOMP) [4].…”

“…Immunization of animals. An immunization schema for Вalb/с mice was chosen according to available literature data and own experience [4][5][6][7][8][9]. The most effective immune response is usually developed following immunization by antigen mixed with Freund's complete adjuvant (FCA) into hind leg pads.…”

Biotechnology for obtaining hybrid positive control samples for immunoassay for detecting antibodies against Chlamydia trachomatis

“…As already noted, in the case of producing ELISA kits intended to detect specific IgG antibodies, there is no problem in obtaining positive controls, since immunoglobulins of the IgG class are the major protective antibodies in the serum of infected individuals and convulsants in relatively high titres. At the same time, for the large-scale production of immunoassay kits designed for detection of IgA and IgM antibodies, there are limitations on the availability of large amounts of starting material -IgA and IgM positive sera (Galkin & Gorshunov, 2014;Galkin et al, 2015). An adjuvant that solves such a disorder is the usage of hybrid positive controls, including: universal synthetic positive control for IgM capture ELISA -a conjugate of normal human IgM and McAbs to the horseradish peroxidase, as well as synthetic positive control for indirect ELISA, which is designed for detection of IgM and IgA antibodies to Ch.…”