Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli

Abstract

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38-and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn grain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A 405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.

Inhibition of aflatoxin B1production ofAspergillus flavus, isolated from soybean seeds by certain natural plant products

“…Aflatoxins are potent toxic, carcinogenic, mutagenic, immunosuppressive and teratogenic agents produced as secondary metabolites by A. flavus and A. parasiticus (Shapira et al. 1997).…”

Métodos imunológicos para a detecção de fungos toxigênicos e micotoxinas em grãos, alimentos e rações

“…Anticorpos produzidos contra duas proteínas quiméricas recombinantes produzidas em Escherichia coli, resultantes dos genes ver-1 e apa-2 de A. parasiticus, que estão envolvidos na biossíntese de aflatoxinas, apresentaram potencial para serem utilizados em métodos imunológicos para detecção de fungos aflatoxigênicos. Esses anticorpos reagiram mais fortemente com antígeno de A. parasiticus e A. flavus em relação a antígenos de outros fungos quando analisados por ELISA, enquanto os anticorpos produzidos contra o filtrado do cultivo de A. parasiticus reagiram com todas as espécies de Aspergillus e Penicillium, indicando a maior especificidade dos anticorpos produzidos contra as proteínas quiméricas envolvidas na biossíntese de aflatoxinas 44 .…”