Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau

Specific and total serum immunoglobulins were extracted by immunoaffinity, mannan-binding protein and Protein A affinity chromatography from southern bluefin tuna (Thunnus maccoyii Castelnau) immunised with rabbit IgG, and fr om non-immunised southern bluefin tuna. SDS-PAGE in 10% reducing gels revea led two heavy chains with molecular weights of approximately 74.6 +/- 1.3 k Da and 71.2 +/- 0.9 kDa, and two light chains with molecular weights of app roximately 29 +/- 1.2 kDa and 28 +/- 1.0 kDa. Under non-reducing, but denat uring, conditions in 4% and 5% SDS-PAGE gels, a high molecular weight and a low molecular weight fraction were demonstrated. By get filtration using S ephacryl HR 300 a molecular weight of 845 kDa, consistent with a tetramer, was obtained for the high molecular weight fraction, and a molecular weight of 168 kDa, consistent with a monomer, was obtained for the low molecular weight fraction. The extinction coefficient at A(280) for the purified immu noglobulin (Ig) was determined to be 1.24. Tuna alpha -rabbit IgG Ig was re active with all non-reduced mammalian IgG antigens tested, suggesting that common conformational antigenic determinants were recognised. (C) 2001 Acad emic Press.