Isolation and partial characterization of dogfish (Scyliorhinus canicula) antibody

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Abstract
A humoral immune response was elicited in adult dogfish by intramuscular injection of keyhole limpet haemocyanin (KLH). The antibody raised to this antigen was measured by an agglutination technique. The antibody-containing fraction of the serum was isolated in a two-step procedure utilizing gel nitration on Sepharose 6B and slab gel electrophoresis. A rabbit antiserum was then raised to the purified immunoglobulin. Further gel filtration studies revealed that the anti-KLH agglutinating activity was restricted to a highmolecular-weight serum fraction (approximately 800000) in fish immunized for up to 18 months. No anti-KLH precipitating activity was detected in fish at any stage of immunization; however immunodiffusion studies with the rabbit antiserum raised against dogfish antibody confirmed the existence of an 800000 molecular weight form and also a lighter species weighing 160000 daltons in both normal and immunized fish. The antigenic specificity of this latter molecule was not demonstrated. Immunoelectrophoretic analysis of the antibody revealed a neutral/slight anodic mobility, and after reduction and alkylation the presence of two polypeptide chains weighing 18200 and 75 860 daltons was demonstrated by thin-layer gel chromatography. These physicochemical characteristics make dogfish antibody analogous to mammalian immunoglobulin M (IgM).