Development of Monoclonal Antibody-Based Sensitive Sandwich ELISA for the Detection of Antinutritional Factor Cowpea Trypsin Inhibitor

Trypsin inhibitor in cowpea Vigna unguiculata (CpTI) can be classified as the Bowman-Birk serine protease inhibitor and has been reported to be one of the antinutritional factors. Genes encoding these proteins have become an option to obtain insect-resistant transgenic plants. Monoclonal antibodies (mAbs) were generated using purified trypsin inhibitors from cowpea seeds to immunize the mice. One clone of these antibodies, named mAb 3D6, was selected to conjugate with horseradish peroxidase (HRP) and then used in a sandwich enzyme-linked immunosorbent assay (ELISA) in which the coating antibody was an anti-CpTI polyclonal antibody. The limit of detection and working range of the assay were 0.21 and 1-100 ng/mL, respectively. Western blotting of cowpea seed crude extracts and purified CpTI protein was also established employing mAb 3D6 and HRP-labeled goat anti-mouse antibody. The results of ELISA and Western blotting suggest that mAb 3D6 has application values for studies of CpTI in cowpea and cpti-transgenic plants.