Performance and Applications of Polyclonal Antipeptide Antibodies Specific for the Enterococcal Bacteriocin Enterocin P

Polyclonal antibodies with specificity for enterocin P (EntP) have been generated by immunization of rabbits with two chemically synthesized N-terminal peptides (P1 and P2) and a C-terminal peptide (P3) of this bacteriocin conjugated to the carrier protein KLH. The sensitivity and specificity of the peptide−KLH-generated antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect (CI)-ELISA. The NCI-ELISA but not the CI-ELISA was valuable for detecting the existence of EntP specific antibodies in the sera of the P2−KLH and P3−KLH immunized animals and to detect and quantify the EntP in the supernatant of producer strains. The anti-P2−KLH sera cross-reacted with the supernatant of a strain producer of sakacin A, a bacteriocin closely related to EntP. Immunoaffinity chromatography columns with anti-P2−KLH or anti-P3−KLH immunoglobulins retained the EntP from the supernatant of the producer strain. Western blotting of EntP with the anti-P2−KLH-generated antibodies suggests that purified EntP tends to the formation of aggregates with no antimicrobial activity. Monitoring the purification of EntP with antipeptide antibodies suggests that while the performance of the evaluated purification procedures would be reasonably acceptable in terms of recovery of the antimicrobial activity of the bacteriocin, their yield is far from attractive in terms of recovery of the initial concentration of enterocin P. Keywords: Enterococci; bacteriocins; enterocin P; antipeptide antibodies; ELISA