Production and utilization of polyclonal antibodies against nisin in an ELISA and for immuno-location of nisin in producing and sensitive bacterial strains

Specific nisin polyclonal antibodies (PAb) were produced in rabbits using nisin Z produced by Lactococcus lactis subsp. lactis biovar diacetylactis UL 719. Antisera were obtained from white female New Zealand rabbits that were first immunized with a nisin Z-keyhole limpet haemocyanin conjugate and boosted with free nisin Z. Nisin-specific PAb were purified by affinity chromatography with a yield of 15 mg specific antinisin 100 ml-1 serum. The detection limit of the ELISA test for nisin Z was 0.75 ng ml-1 in buffer but was 1.7 and 3.5 ng ml-1 in milk and complex media broth spiked (5, 10, 20 microg ml-1) with nisin Z, respectively. In nisin Z-spiked samples, the average concentration was between 90 and 107% of actual added amount. In contrast, when the bioassay (microtitration method) was used, only 50-63% of nisin Z biological activity could be detected. In addition, the affinity-purified nisin PAb, antirabbit IgG gold conjugate and transmission electron microscopy were successfully used to locate nisin Z on producing cells and to observe its bactericidal effects against sensitive cells.