Construction and characterization of single chain variable fragment-alkaline phosphatase for rapid detection of aflatoxin B1 in an ELISA-based assay

Aflatoxins are metabolites produced by Aspergillus spp. And can be found as contaminants in various food and agricultural products. A specific antibody is needed for the development of serological method, such as Enzyme-linked immunoserbent assay (ELISA), as a screening process to determine toxin contamination. In this study, nine clones of specific single chain variable fragments (scFV) were selected from naive mouse phage display scFV library and their reactivity with aflatoxins were determined. The experiments were conducted in the Serology and Diagnostic Laboratory, Center of Agricultural Biotechnology, Kasetsart University from 2012-2013. The scFV gene from recombinant phagemid clone 22A12 (scFV-22A12 gene), which gave the strongest reaction, was selected for further investigation of aflatoxin analysis. The recombinant protein product was 30k Da. Concurrently, an alkaline phosphatase (AP) gene was amplified from Escherichia coli strain HB2151. The scFv-22A12 and the AP genes were ligated into pCANTAB-SE phagemid and transformed into E. coli TG1 and HB2151 to produce phage scFv-AP and soluble scFv-AP, respectively. Comparison on the efficiency of Phage scFv, Phage scFv-AP and soluble scFv-AP to whole molecule antibody for detecting AFB1 was performed by ELISA. The results showed that the soluble scFv-AP gave highest reactivity and in accordance with those obtained from the whole molecule antibody. Cross reactivity with other aflatoxins (B2, G1 and G2) was reported to be 38.63%, 21.24% and 9.64%, respectively, when using soluble scFv-AP to analyze ground samples of corn and groundnut spiked with 100 ug/kg of AFB1, acceptable results were obtained with 87.02 and 94.41% recovery, respectively. Analysis of the certified reference material (TMAF No.2 and TMAF No.3) showed comparable results with those analyzed through high performance liquid chromatography.