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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 IIa bacteriocins

Departamento de Nutrición y Bromatología III, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain¹

Author for correspondence: Pablo E. Hernández. Tel: +34 91 3943752. Fax: +34 91 3943743. e-mail: ehernan{at}eucmax.sim.ucm.es

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 keyhole limpet haemocyanin (KLH). The PH1 fragment holds a highly conserved amino acid sequence with closely related Class IIa bacteriocins. The sensitivity and specificity of the PH1–KLH-generated rabbit polyclonal antibodies were evaluated by the development of various ELISAs, such as a non-competitive 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 valuable for detecting the existence of PedA1-specific antibodies in the sera of immunized rabbits. The limit of detection of PedA1 in MRS medium was found to be 0·5 µg ml^-1 in NCI-ELISA, while CI-ELISA on plates coated with purified PedA1 increased the affinity of the PH1–KLH-generated antibodies for PedA1; the limit of detection of PedA1 was less than 0·01 µg ml^-1 and 50% binding inhibition was achieved with 0·1 µg PedA1 ml^-1. Similarly, the limits of detection of PedA1 in MRS medium were found to be 5 µg ml^-1 by protein slot-blotting and 0·01 µg ml^-1 by Western blotting. Most importantly, PH1–KLH-generated polyclonal antibodies detected the presence of PedA1 in the supernatants of the producing strains of Pediococcus acidilactici 347, Z102, A172, X13 and P20, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing closely related or different bacteriocins. The approaches taken for the selection of the bacteriocin peptide fragment, the generation of 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.

Keywords: pediocin PA-1, bacteriocin, immunodetection, lactic acid bacteria

Abbreviations: ADT, agar diffusion test; CB, coating buffer; CD-, CI-, NCI-and S-ELISA, competitive direct, competitive indirect, non-competitive indirect and sandwich ELISA; KLH, keyhole limpet haemocyanin; LAB, lactic acid bacteria; MPA, microtitre plate assay; OA, ovalbumin; PedA1, pediocin PA-1; TMB, 3,3',5,5'-tetramethylbenzidine

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