Generation of Polyclonal Antibodies of Predetermined Specificity against Pediocin PA-1

ABSTRACT Polyclonal antibodies of predetermined specificity for pediocin PA-1 (pedA1) have been generated by immunization of rabbits with a chemically synthesized C-terminal fragment of this bacteriocin (PH2) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of the PH2-KLH-generated antibodies were evaluated by the development of various enzyme-linked immunosorbent assays (ELISAs)—a noncompetitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), and a competitive direct ELISA (CD-ELISA)—and by immunodotting. All immunoassays indicated the existence of pedA1-specific 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 μg/ml by immunodotting and 1 μg/ml in the NCI-ELISA. However, the CI-ELISA enhanced the limit of detection of pedA1 to 0.025 μg/ml, while the amount of free pedA1 required for 50% binding inhibition was 10 μg/ml. Moreover, the CD-ELISA increased the affinity of the PH2-KLH-generated antibodies for pedA1; the limit of detection of pedA1 was less than 0.025 μg/ml, and the 50% binding inhibition value was reduced to 0.5 μg of pedA1/ml. All immunoassays and the slot dot assay 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 taken 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 food industry.

Polyclonal antibodies with predetermined specificity against bovine αs1-casein: application to the detection of bovine milk in ovine milk and cheese

SummaryComparing the primary sequences of bovine and ovine milk proteins, some short peptide fragments are cow-specific, in particular the 141–148 fragment of bovine αs1-casein, which is deleted in its ovine counterpart. The 140–149 peptide was chemically synthesized on a solid phase matrix and directly used as an immunogen to produce polyclonal monospecific antibodies in rabbits. These antibodies recognized this fragment both on the peptidyl resin and in the native protein. They appeared to be monospecific, since no antigen–antibody complex was formed with homologous ovine or caprine proteins. Subsequently, a competitive enzyme-linked immuno-sorbent assay was successfully developed for the detection of defined amounts of cows' milk in sheep's milk from 0·125 to 64% (v/v) and in cheese from 0·5 to 25% (v/v) that was not influenced by heat treatment of milk or the degree of ripening of cheese.