Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis (Bt) CrylB toxin

摘要

Single chain variable fragment antibody (scFv) is capable of binding its target antigens and is one of the most popular recombinant antibodies format for many applications. In this study, a large human synthetic phage displayed library (Tomlinson J) was employed to generate scFvs against CrylB toxin by affinity panning. After four rounds of panning, six monoclonal phage particles capable of binding with the CrylB were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Two of the identified novel anti-CrylB scFvs, namely H9 and B12, were expressed in Escherichia coli HB2151 and purified by Ni metal ion affinity chromatography. Sodium dodecyl sulfate poly-acrylamine gel electrophoresis (SDS-PAGE) indicated that the relative molecular mass of scFv was estimated at 30 kDa. The purified scFv-H9 was used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-EUSA) for CrylB toxin. The linear range of detection for standards in this ic-ELISA was approximately 0.19-1.1 μg mL~(-1) and 50% inhibition of control (IC_(50)) was 0.84 μg mL~(-1) for CrylB. The affinity of scfv-H9 was (1.95 ± 0.12) x 10~7 M~(-1) and showed cross-reactivity with CrylAb toxin and CrylAc toxin (8.53% and 7.58%, respectively), higher cross-reactivity (12.8%) with Cry1C toxin. The average recoveries of CrylB toxin from spiked leaf and rice samples were in the range 89.5-96.4%, and 88.5-95.6%, respectively, with a coefficient of variation (C.V) less than 6.0%. These results showed promising applications of scfv-H9 for detecting CrylB toxin in agricultural and environmental samples.