Development of Monoclonal Antibody-Based Sensitive Sandwich ELISA for the Detection of Antinutritional Factor Cowpea Try
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Development of Monoclonal Antibody-Based Sensitive Sandwich ELISA for the Detection of Antinutritional Factor Cowpea Trypsin Inhibitor G. Y. Tan & T. G. Nan & W. Gao & Q. X. Li & J. J. Cui & B. M. Wang
Received: 30 December 2011 / Accepted: 7 June 2012 / Published online: 30 June 2012 # Springer Science+Business Media, LLC 2012
Abstract 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 antiCpTI 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 cptitransgenic plants. T. G. Nan contributed equally to this work. G. Y. Tan : T. G. Nan : W. Gao : B. M. Wang (*) College of Agricultural and Biotechnology, China Agricultural University, Beijing 100193, China e-mail: [email protected] Q. X. Li Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA J. J. Cui (*) Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang, Henan 455000, China e-mail: [email protected]
Keywords Cowpea trypsin inhibitor . GMO . Monoclonal antibody . Sandwich ELISA
Introduction Trypsin inhibitor in the cowpea Vigna unguiculata (CpTI), one of the Bowman-Birk inhibitors with a molecular weight (MW) of 12–16 kDa, was first reported in 1947 (Borchers et al. 1947). It presents as a mixture of monomer, dimer, and tetramer, of which the dimer often being dominant (Qi et al. 2005; Rao et al. 1999, 2007; Barbosa et al. 2007). Due to the characteristic that trypsin and chymotrypsin inhibition in the digestive process of the ingesting monogastric animal, CpTI was defined as antinutritional factor. Pusztai et al. (1992) reported CpTI provoked moderate reduction in weight gain and depression of net protein utilization in rats. Grant et al. (1993) also found evident pancreatic enlargement in rats fed with diets containing raw soybeans or cowpeas, and they considered the change was mediated by the trypsin inhibitors. When CpTI inhibits insect proteases involved in digestive processes, such inhibition can result in a depletion of essential amino acids required for growth, which in turn causes the larvae to die (Angharad et al. 1979). For the function as a natural agent against herbivorous insects, CpTI has received co
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