Establishment of a simple and quantitative immunospot assay for detecting anti-type II collagen antibody using an...

Antibodies to type II collagen (col II) have been detected in patients with rheumatoid arthritis and in animal models of collagen induced arthritis. Here, we describe a novel method to detect anti-col II antibodies using an immunospot assay with an infrared fluorescence imaging system. This method showed very high sensitivity and specificity, and was simple, with low background levels. It also showed higher reproducibility and linearity, with a dynamic range of approximately 500-fold, than the conventional immunospot assay with enhanced chemiluminescence detection. Using this method we were able to demonstrate the antibody affinity maturation process in mice immunized with col II. In these immunized mice, although cross-reactive antibodies reacting with other collagen species were detected in earlier stages of immunization, the titers of cross-reactive antibodies rapidly diminished after the antigen boost, concomitantly with the elevation of the anti-col II antibody. The method and its possible applications are discussed.