Monoclonal antibodies differentially reactive with native and reductively modified Bowman-Birk protease inhibitor

Bowman-Birk protease inhibitor (BBI) is a potent anticarcinogen that suppresses malignant transformation at nanomolar concentrations. Small amounts of BBI in its native form can be measured by immunoassay using specific monoclonal antibodies (MAbs); however, the MAbs currently available are not capable of detecting BBI metabolites in human body fluids. To develop new reagents for the study of BBI exposure and pharmacokinetics, we produced four MAbs, designated 3B6, 3E3, 4H8 and 5G2, from hybridomas derived from a mouse immunized with reductively modified BBI. The epitopes recognized by the four MAbs were characterized using BBI in its native form or modified by different methods. MAb 3B6 reacted with native BBI. Partial reduction of BBI with 720 Gy of gamma radiation in an oxygen-free solution of 100 mM formate increased the reactivity of BBI with 3B6; however, extensive reduction of BBI with 100 mM DL-dithiothreitol (DTT) completely abolished this antigenic reactivity. In contrast, the other three MAbs reacted with BBI molecules that had been reduced either with 720 Gy of radiation in formate solution or with DTT. Alkylation of the radiochemically reduced BBI with N-ethylmaleimide further increased the reactivity of BBI with 3E3, 4H8 and 5G2, possibly by preventing the formation of new disulfide bonds within the BBI molecules. The binding of 4H8 and 5G2 to BBI antigen was inhibited by the binding of 3E3, and vice versa. Thus, the epitopes recognized by 3E3, 4H8 and 5G2 are probably located close to one another on the reduced BBI molecules. These three MAbs were able to react with BBI metabolites in urine samples collected from volunteers after oral administration of BBI. The ability of these MAbs to detect BBI metabolites indicates that BBI may be reductively modified in vivo and these MAbs may be useful reagents for monitoring the uptake of BBI into human tissues in cancer chemoprevention studies with BBI.