Development of a Monoclonal Antibody Against Tursiops truncatus IgG For Use as a Reagent in a Diagnostic ELISA
IAAAM Archive
Nicole A. Lilly; Rhonda A. Patterson; Bobby L. Middlebrooks
University of Southern Mississippi, Department of Biological Sciences, Hattiesburg, MS, USA


IgG was purified from pooled dolphin sera by affinity chromatography using Protein G Sepharose 4 Fast Flow (Pharmacia). A set of four to five week old female BALB/c mice were immunized with 100 ug each of purified IgG. At five weeks, the animals were sacrificed and their spleens aseptically removed. The spleen cells were removed for fusion with the myeloma cell line CRL 1580 (ATCC). Myeloma cells were added to spleen cells at a 5:1 myeloma/spleen cell ratio, and the cells in the mixed suspension were fused using a 50% solution of polyethylene glycol (1450 molecular weight) at 37°C. On day ten screening for hybrids secreting antibodies was performed using an enzyme linked immunosorbent assay (ELISA). Wells yielding absorbance (405 nm) readings above 0.2 were considered positive for production of immunoglobulin. The ELISA was repeated and the positive wells were checked for growth. Wells containing viable cells were removed for limiting dilution cloning and expansion of the individual clones. To date, the class and specificity of one of these clones has been determined. A mouse isotyping strip (Serotec, Inc.) indicated that the monoclonal antibody is IgA with kappa light chains. Based on these findings, concentrated cell supernatant was applied to a Jacalin column, which is known to bind to carbohydrate moieties on IgA molecules, and analysis of the column elutions confirmed the test strip results. Further confirmation that the immunoglobulin was IgA came from molecular weight analysis via SDS-PAGE and positive binding in a Western blot probed with antisera specific for mouse IgA. To determine the specificity of the monoclonal antibody produced, an appropriately designed ELISA and a Western blot were performed. The results indicated the antibody produced by the hybridoma was specific for dolphin IgG heavy chain. This monoclonal antibody was then used in a ELISA designed to quantitate the levels of IgG in a set of dolphin serum samples.

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Nicole A. Lilly

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