Antibodies to a synthetic 1-9-N-terminal amino acid fragment of mature pediocin PA-1: sensitivity and specificity for pediocin PA-1 and cross-reactivity against Class lla bacteriocins

Polyclonal antibodies specific for pediocin PA-1 (PedA1) were generated by immunization of rabbits with a chemically synthesized 1-9-N-terminal amino acid fragment of this bacteriocin (PH1) conjugated to the carrier protein k eyhole limpet haemocyanin (KLH). The PH1 fragment holds a highly conserved amino acid sequence with closely related Class IIa bacteriocins. The sensit ivity and specificity of the PH1-KLH-generated rabbit polyclonal antibodies were evaluated by the development of various ELISAs, such as a non-competi tive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), a competitive direct ELISA (CD-ELISA) and a sandwich ELISA (S-ELISA), and by protein slot-blotting and Western blotting. NCI- and CI-ELISA were valuabl e for detecting the existence of PedA1-specific antibodies in the sera of i mmunized rabbits. The limit of detection of PedA1 in MRS medium was found t o be 0.5 mu g ml(-1) in NCI-ELISA, while CI-ELISA on plates coated with pur ified PedA1 increased the affinity of the PH1-KLH-generated antibodies for PedA1; the limit of detection of PedA1 was less than 0.01 mu g ml(-1) and 5 0% binding inhibition was achieved with 0.1 mu g PedA1 ml(-1). Similarly, t he limits of detection of PedA1 in MRS medium were found to be 5 mu g ml(-1 ) by protein slot-blotting and 0.01 mu g ml(-1) by Western blotting. Most i mportantly, PH1-KLH-generated polyclonal antibodies detected the presence o f PedA1 in the supernatants of the producing strains of Pediococcus acidila ctici 347, Z102, A172, X13 and P20, with no reactivity or negligible immuno reactivity with the supernatants of other lactic acid bacteria producing or not producing closely related or different bacteriocins. The approaches ta ken for the selection of the bacteriocin peptide fragment, the generation o f antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of adequate specificity for other bacteriocins of interest in the food industry.