Improvement of immunoglobulin M capture immunoassay specificity: toxoplasma antibody detection method as a model

Abstract

In the Toxoplasma gondii immunoglobulin M (IgM) capture fluorometric enzyme immunoassay used as a model, nonspecific responses due to the binding of human IgM to horseradish peroxidase (HRP) conjugates were observed despite the removal of the Fc portion of the immunoglobulin. This interaction may be mediated through the binding of human IgM to the HRP moiety of the conjugate. Addition of polymerized HRP into the reaction mixture reduced nonspecific signals in the majority of low false-positive serum reactions. Other plausible sites of interaction are conserved epitopes of mouse immunoglobulins presenting antigenic similarities with the allotopes of other species. Fragmentation of mouse antimicrobial IgG to Fab' and selection of proper conjugation procedure improved assay specificity.

FIG. 1

Reactivities of samples 1 to 16 from Arhangelsk (Russia) and sample Ang from a laboratory member by capture T. gondii FEIA. The same reaction conditions were employed throughout the experiments. (A) Reactivities of samples with anti-human IgG capture antibody. (B) Reactivities of samples with anti-human IgA capture antibody. (C to E) Reactivities of samples with anti-human IgM capture antibody. The conjugates and concentrations are indicated in Table 1. The samples were tested in the presence (solid bars) and in the absence (open bars) of T. gondii antigen. Sample 5 was not available for all experiments. The negative, borderline, low-positive, and positive T. gondii IgM controls are marked N, B, L, and P, respectively.