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

Abstract

The enzyme-linked immunosorbent assay (ELISA) is the most informative and versatile method of serological diagnostics. The possibility of detection by ELISA of specific antibodies of different classes allow one to differentiate primary infectious processes and their remission, exacerbation and chronic disease (holding of differential diagnosis). This approach is implemented in the methodology for evaluation of patients for the presence of humoral immune response against TORCH-infections pathogens (toxoplasmosis, rubella, cytomegalovirus, herpes simplex viruses infections, and some others). Therefore, testing for the presence of specific IgG and IgM antibodies against TORCH-infection pathogens in blood serum is an important element of mother and child protection. The essential problem in the production of IgM-capture ELISA diagnostic kits is obtaining positive control. The classic version of positive control is human blood serum (plasma) containing specific antibodies. But specific IgM-positive sera are insignificant raw material. This fact can significantly limit the production of diagnostic kits, especially in case of large-scale manufacture. We have suggested a methodological approach to the use of synthetic positive controls in IgM-capture ELISA kits based on conjugate of normal human IgM and monoclonal antibodies against horseradish peroxidase. It was found that it is possible to realize such a task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), reductive amination-mediated conjugation (by sodium periodate) and glutaraldehyde-mediated conjugation. It was found that conjugates of normal human IgM and monoclonal antibodies against horseradish peroxidase obtained using NHS ester-mediated maleimide conjugation and periodate method are homogeneous in molecular weight, whereas conjugate synthesized by glutaraldehyde method comprises at least three types of biopolymers with close molecular weight. It was found that synthetic positive control obtained by different methods are characterized by higher titre compared to IgM-positive high-titre serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics. We have suggested a methodological approach to the 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 is 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 control obtained by different methods are characterized by higher titre compared to IgM- and IgA-positive high-titre serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics, both at the release time and after a week’s storage at 37 °C.

Keywords

• elisa; human immunoglobulins; synthetic control; chlamydia trachomatis; conjugation