Monoclonal Antibodies Against Soybean Bowman-Birk Inhibitor Recognize the Protease-Reactive Loops

Abstract

Monoclonal antibodies against soybean Bowman-Birk protease inhibitor (BBI) have been generated and used to detect and quantify BBI in foods, soybean germplasm, and animal tissues and fluids. The purpose of this study was to determine the recognition sites of two monoclonal antibodies to BBI (mAb 238 and mAb 217) in relation to the protease-inhibitory sites of BBI. The results showed that (1) the binding of mAb 238 can be blocked by trypsin and that of mAb 217 by chymotrypsin; (2) the trypsin or chymotrypsin inhibitory activities of BBI are blocked by mAb 238 or mAb 217, respectively; and (3) mAb 238 failed to recognize a tryptic loop mutant BBI variant and mAb 217 was unable to bind a chymotryptic loop mutant BBI variant. These findings demonstrate that the epitopes recognized by mAb 238 and mAb 217 reside, at least in part, in the tryptic and chymotryptic loops of BBI, respectively.

Probing the Soybean Bowman–Birk Inhibitor Using Recombinant Antibody Fragments

“…We started from the mRNA of the anti-BBI cell line secreting the monoclonal mAb 238 previously characterized. 16,20,22 Then, monovalent (singlechain Fv, scFv) and multivalent (single-chain diabody, scDb) anti-BBI recombinant antibody fragments were developed using the principles of genetic engineering and protein design. Figure 1a contains the general strategy we used to clone and obtain each antibody construct as single recombinant proteins.…”

“…Surprisingly, the advancement of recombinant antibody technology is under-represented in food research and industry, an area where there is a need for improved analytical and purification methodologies. 22,31 Just as soy consumption patterns vary greatly across the world, so too do cancer incidences. An inverse relationship between soybean intake and cancer incidence has been reported, with Asian countries where soy forms an important part of the diet, such as Japan, displaying the lowest incidences of cancer.…”

“…Its paratope targets the tryptic loop (conformational epitope) of BBI. More interestingly, its reactivity with other soybean-derived protease inhibitors (i.e., Kunitz) is very low 22 and nonexistent with BBI derived from other sources. Therefore, mAb 238 seems to be the ideal scaffold on which to develop antibody-based affinity purification methodology.…”

Bowman-Birk and Kunitz Protease Inhibitors among Antinutrients and Bioactives Modified by Germination and Hydrolysis in Brazilian Soybean Cultivar BRS 133

“…It is important to mention that in this study, both trypsin inhibitory domains present in the BBI isoinhibitor were recognized by the antibody used, based on a previous study on the reactivity of the same BBI antibody (mAb 238). 26 Previous studies have shown that germination alone had an effect on trypsin inhibitory activity in soybeans. Mostafa and Rahma 27 found that germination of soybean seeds for 6 days reduced trypsin inhibitory activity by 32%, while Bau et al 28 showed that KTI and BBI degradation was enhanced if allowed to germinate for more than 4 days.…”

Isolation of Bowman-Birk-Inhibitor from soybean extracts using novel peptide probes and high gradient magnetic separation

“…Mouse monoclonal antibodies against BBI have been raised laboriously using the hybridoma approach, allowing quantitative determination of BBI in soy protein mixtures (Brandon, Bates et al 1989;Seerama & Gowda, 1997;Mao et al, 2005) but the lack of probes and purification methodologies has impeded progress. To date, tedious and time consuming, multi-step chromatographic procedures have been the convention for BBI purification.…”