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 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 gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) 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.
Shapira
R
R
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Paster
N
N
Menasherov
M
M
Eyal
O
O
Mett
A
A
Meiron
T
T
Kuttin
E
E
Salomon
R
R
D016604:Aflatoxin B1 / Q000032:analysis / Q000096:biosynthesis*; D000818:Animals; D000908:Antibodies, Fungal / Q000276:immunology*; D001230:Aspergillus / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology*
D000908:Antibodies, Fungal; D011993:Recombinant Fusion Proteins; D016604:Aflatoxin B1