DEVELOPMENT OF POLYCLONAL ANTIBODIES FOR DETECTION OF AFLATOXIGENIC MOLDS INVOLVING CULTURE FILTRATE AND CHIMERIC PROTEINS EXPRESSED IN ESCHERICHIA-COLI

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strai n of Aspergillus parasiticus by using two different sources for antibo dy elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli a s separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, an d each was cloned into the E. coli expression vector pGEX2T. Upon indu ction, the bacteria overexpressed 38- and 33-kDa chimeric proteins cor responding to the N-terminal domains of the genes ver-1 and apa-2, res pectively, The chimeric proteins were isolated and affinity purified f or use as antigens. The specificity of the raised antibodies was exami ned by enzyme-linked immunosorbent assay (ELISA). The PAbs raised agai nst the culture filtrate reacted with all the species of Aspergillus a nd Penicillium tested but not with Fusarium species or corn grain, How ever, the PAbs elicited against the chimeric proteins were highly spec ific, showing significantly higher ELISA absorbance values (A(405)) ag ainst A. parasiticus and A. flavus than against the other fungi tested and the corn grain, The approach of utilizing gene products associate d with aflatoxin biosynthesis for antibody production therefore appear s to be feasible. Such a multiantibody system combined with the PCR te chnique, could provide a useful tool for the rapid, sensitive, and acc urate detection of aflatoxin producers present in grains and foods.