DEVELOPMENT OF POLYCLONAL ANTIBODIES FOR DETECTION OF AFLATOXIGENIC MOLDS INVOLVING CULTURE FILTRATE AND CHIMERIC PROTEINS EXPRESSED IN ESCHERICHIA-COLI
R. Shapira et al., DEVELOPMENT OF POLYCLONAL ANTIBODIES FOR DETECTION OF AFLATOXIGENIC MOLDS INVOLVING CULTURE FILTRATE AND CHIMERIC PROTEINS EXPRESSED IN ESCHERICHIA-COLI, Applied and environmental microbiology, 63(3), 1997, pp. 990-995
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.