An antibody class capture technique is used. This involves the dilution of the sample and the simultaneous reaction of IgM in the diluted sample with biotinylated mouse monoclonal antihuman IgM antibody. The immune complex is captured by streptavidin on the wells. Unbound materials are removed by washing. Horseradish peroxidase (HRP)-labeled mouse monoclonal antihepatitis A virus (anti-HAV) IgM, which has been complexed with recombinant HAV antigen (conjugate), is then captured by anti-HAV-specific IgM bound to the wells. Unbound material is removed by washing.
The bound HRP conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent, is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent increases the level and duration of the light produced. The light signals are read by the VITROS ECi system. The amount of HRP conjugate is indicative of the concentration of anti-HAV IgM present in the sample.(Package insert: VITROS Anti-HAV IgM Reagent Pack, no. GEM1235A, version 3.0, Ortho-Clinical Diagnostics, Inc. Rochester, NY 14626-5101, 8/18/2009)