Isolation from nurse shark serum of immune 7s antibodies with two different molecular weight H-chains

Abstract

We have isolated 7S antibodies from the sera of nurse sharks hyperimmunized with bovine gamma globulin (BGG). The procedures employed in the purification were affinity column chromatography. BGG linked to Sepharose-4B and DE 52 column chromatography. The antibodies isolated by these procedures were reactive with BGG and pure by the criteria of Ouchterlony. immunoelectrophoresis and polyacrylamide gel electrophoresis (PAGE). Reductive cleavage of the 7S anti-BGG antibodies with dithiothreitol (DTT) produced predominantly 50,000 mol. wt H-chains and to a lesser degree. 72.000 mol. wt H-chains on SDS-PAGE. The L-chains were of 23.000 mol. wt. Natural 19S and 7S immunoglobulins were purified from non-immune shark sera by low ionic strength precipitation (Jensen, 1969). polyethylene glycol precipitation, DE 52 column chromatography and Concanavalin A-Sepharose affinity column chromatography. Reductive cleavage of the natural 19S and 7S immunoglobulins with DTT and analysis by SDS-PAGE revealed only 72,000 mol. wt H-chains and 23.000 mol. wt L-chains. Experimental evidence indicated that two types of 7S anti-BGG antibodies were engendered after immunization, one with H-chains of 50,000 mol. wt 7S(IgH 50) and another with H-chains of 72.000 mol. wt 7S (IgM). The IgH 50 is present in non-immune sera at low concentrations. A sensitive and specific procedure was developed to detect the IgH 5 immunoglobulins in non-immune sera. It involved: fluorescent labeling of the shark's immunoglobulins: specific precipitation of the labeled immunoglobulins, and separation and detection of the fluorescent labeled H-chains.