Production and characterization of monoclonal antibodies against norsolorinic acid reductase involved in aflatoxin biosynthesis

Using a partially purified norsolorinic acid reductase (NSR) preparation as the immunogen, monoclonal antibodies (mAbs) against NSR, an enzyme respons ible for the conversion of norsolorinic acid to averantin in the early stag e of aflatoxin biosynthesis, were produced. An ELISA, using partially purif ied NSR as coating antigen and a second antibody-peroxidase conjugate as in dicator, was established for measurement of the antibody titre and enzyme l evel. A total of 12 hybridoma cell lines that produced antibodies against v arious proteins were obtained. HPLC-postcolumn ELISA, ir;immunoblot analysi s and enzyme inhibition study revealed that a mAb elicited in cell line 10D 2 specifically bound with the 43 kDa NSR. Immunoblot and ELISA analyses of extracts from various fungal cultures showed that the 10D2 mAb was highly s pecific to the NSR; only culture extracts with positive NSR activities cont ained 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 neutrali zing the NSR activity and has also been used for detection of expression of the gene encoding the enzyme.