Production and Characterization of Monoclonal Antibodies against Norsolorinic Acid Reductase Involved in Aflatoxin Biosynthesis

Abstract

Using a partially purified norsolorinic acid reductase (NSR) preparation as the immunogen, monoclonal antibodies (mAbs) against NSR, an enzyme responsible for the conversion of norsolorinic acid to averantin in the early stage of aflatoxin biosynthesis, were produced. An ELISA, using partially purified NSR as coating antigen and a second antibody-peroxidase conjugate as indicator, was established for measurement of the antibody titre and enzyme level. A total of 12 hybridoma cell lines that produced antibodies against various proteins were obtained. HPLC-postcolumn ELISA, immunoblot analysis and enzyme inhibition study revealed that a mAb elicited in cell line 10D2 specifically bound with the 43 kDa NSR. Immunoblot and ELISA analyses of extracts from various fungal cultures showed that the 10D2 mAb was highly specific to the NSR; only culture extracts with positive NSR activities contained the 43 kDa band and had a positive reaction with the antibody in the ELISA. The mAb produced by 10D2 cell line was capable of partially neutralizing the NSR activity and has also been used for detection of expression of the gene encoding the enzyme.

Keywords:

• Norsolorinic Acid Reductase
• Aspergillus Flavus
• A. Parasiticus
• Aflatoxins
• Biosynthesis
• Antibodies