Evaluation of bio-conjugation methods for obtaining of synthetic positive control for IgM-capture ELISA

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 (differential diagnosis). This approach is implemented in the methodology for evaluation of patients for the presence of humoral immune response to TORCH-infections pathogens (toxoplasmosis, rubella, cytomegalovirus, herpes simplex viruses’ infections, and some others). Therefore, testing for presence of specific IgG and IgM antibodies against TORCH-infections pathogens in blood serum is an important element of motherhood and childhood protection. The essential problem in the production of IgM-capture ELISA diagnostic kits is obtaining of positive control. The classic version of positive control is human blood serum (plasma) containing specific antibodies. But specific IgM-positive sera are insignificant raw materials. This fact can seriously restrict the production of diagnostic kits, especially in the event of large-scale production. We have suggested the methodological approach to using of synthetic positive controls in IgM-capture ELISA kits based on conjugate of normal human IgM and monoclonal antibodies against horseradish peroxidase. It is found that this task can be fulfilled 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 were similar by molecular weight, whereas conjugate synthesized by glutaraldehyde method comprised at least three types of biopolymers with close molecular weight. It was found that synthetic positive control obtained by different methods was characterized by higher titer compared to IgM-positive high-titer serum. However, positive control obtained by NHS ester-mediated maleimide conjugation had the best titration profile characteristics.

Keywords

• elisa
• igm
• positive control
• conjugation