GENERATION OF POLYCLONAL ANTIBODIES OF PREDETERMINED SPECIFICITY AGAINST PEDIOCIN PA-1

Polyclonal antibodies of predetermined specificity for pediocin PA-1 ( pedA1) have been generated by immunization of rabbits with a chemicall y synthesized C-terminal fragment of this bacteriocin (PH2) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivi ty and specificity of the PH2-KLH-generated antibodies were evaluated by the development of various enzyme-linked immunosorbent assays (ELIS As)-a noncompetitive indirect ELISA (NCI-ELISA), a competitive indirec t ELISA (CI-ELISA), and a competitive direct ELISA (CD-ELISA)-and by i mmunodotting. All immunoassays indicated the existence of pedA1-specif ic antibodies with high relative affinities and adequate sensitivities in the sera of immunized animals. The limits of detection of pedA1 in MRS medium (Oxoid Ltd., Basingstoke, United Kingdom) were found to be 2.5 mu g/ml by immunodotting and 1 mu g/ml in the NCI-ELISA. However, the CI-ELISA enhanced the limit of detection of pedA1 to 0.025 mu g/m l, while the amount of free pedA1 required for 50% binding inhibition was 10 mu g/ml. Moreover, the CD-ELISA increased the affinity of the P H2-KLH-generated antibodies for pedA1; the limit of detection of pedA1 was less than 0.025 mu g/ml, and the 50% binding inhibition value was reduced to 0.5 mu g of pedA1/ml. All immunoassays and the slot dot as say detected the presence of pedA1 in the supernatant of the producing strain Pediococcus acidilactici 347, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing different bacteriocins. The approaches take n for the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of predetermined specificity for other bacteriocins of interest in the f ood industry.