Monoclonal antibodies to soybean kunitz trypsin inhibitor and

Abstract

Hybrid cell lines (hybridomas) which produce and secrete monoclonal antibodies with three distinct patterns of recognition are described. The first specificity pattern is defined by antibodies which (1) recognize Kunitz trypsin inhibitor (KTI), one of the principal protease inhibitors found in soybeans, but do not detect the Bowman-Birk inhibitors (BBI), the other major class of protease inhibitors in soybeans; (2) bind to native KTI isoforms a and b but do not react with KTI isoforms a and b which have been denatured by moist heat or alkaline treatment or which have been subjected to disulfide exchange; and (3) do not recognize native KTI isoform c. The second specificity pattern is defined by antibodies that (1) recognize KTI, but do not bind BBI; (2) bind native KTI isoforms a and c, but do not bind strongly to KTI isoforms a and c which have been denatured by moist heat or alkaline treatment or which have been subjected to disulfide exchange; (3) do not bind KTI isoform b; and (4) bind only weakly to KTI when KTI is complexed with trypsin or a similar enzyme. The third specificity pattern is defined by antibodies that (1) recognize KTI, but do not bind BBI; (2) bind equivalently to native KTI isoforms a, b, and c, but do not bind to KTI which has been denatured by moist heat or alkaline treatment or which has been subjected to disulfide exchange; and (3) bind equivalently to uncomplexed KTI and KTI complexed with trypsin or a similar enzyme. Immunoassay methods using the monoclonal antibodies to analyze native KTI specifically in soy-derived foodstuffs and in tissues of soybean plants, to determine isoform content of a sample, and to determine the amount of KTI complexed with trypsin in a sample are described.