Development of specific enzyme-linked immunosorbent antibody assay systems for the detection of chicken immunoglobulins G, M, and A using monoclonal antibodies

The aim of the present study was the development of a sensitive and specific ELISA system for the quantitative and qualitative assay of chicken Ig Isotypes G, M, and A using monoclonal antibodies. Five hybridoma cell lines were developed that synthesized specific antibodies against chicken IgG and three lines each producing specific antibodies against IgM or IgA. Using an immunodiffusion test, the subclasses were determined. Isolation of monoclonal antibodies from ascites was carried out by way of affinity chromatography with protein G sepharose. The purity of the eluates were determined by both SDS-PAGE and HPLC. A Sandwich ELISA was found to be the most suitable technique for the assay. Specificity testing was carried out by Western blotting. An epitope analysis was also carried out. By variation of the single steps concerning incubation times, quantities, and concentrations of the substances to be applied, the whole procedure was optimized. Assay limits for individual Ig isotypes were determined. The limits were 20 ng/mL for IgG, 80 ng/mL for IgM, and 160 ng/mL for IgA