GENERATION OF POLYCLONAL ANTIBODIES AGAINST NISIN - IMMUNIZATION STRATEGIES AND IMMUNOASSAY DEVELOPMENT

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin n isin A (nisA) have been produced by immunization with nisA-cholera tox in and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immu nized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-s pecific antibodies with high relative affinities and much-increased se nsitivities. nisA antibodies could also be readily mass produced in le ss than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A co mpetitive direct enzyme-linked immunosorbent assay (ELISA) whereby nis A-horseradish peroxidase and free nisA competed for antibody binding w as devised, The detection limit for nisA in the competitive direct ELI SA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, whi le the amount of free nisA required for 50% antibody binding inhibitio n ranged from 0.3 to 5 mu g/ml. Both antisera and ascites polyclonal a ntibodies cross-reacted with nisZ either in the supernatant of a produ cer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies s hould find a wide usage for nisA ELISA analysis in foods and other mat rices.