Performance and Applications of Polyclonal Antipeptide Antibodies Specific for the Enterococcal Bacteriocin Enterocin P
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- 作者：Jorge Gutié ; rrez ; Raquel Criado ; Rosanna Citti ; Marí ; a Martí ; n ; Carmen Herranz ; Marí ; a F. Ferná ; ndez ; Luis M. Cintas ; and Pablo E. Herná ; ndez
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2004
- 出版时间：April 21, 2004
- 年：2004
- 卷：52
- 期：8
- 页码：2247 - 2255
- 全文大小：116K
- 年卷期：v.52,no.8(April 21, 2004)
- ISSN：1520-5118
文摘
Polyclonal antibodies with specificity for enterocin P (EntP) have been generated by immunization ofrabbits 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 thepeptide-KLH-generated antibodies were evaluated by a noncompetitive indirect enzyme-linkedimmunosorbent assay (NCI-ELISA) and a competitive indirect (CI)-ELISA. The NCI-ELISA but notthe CI-ELISA was valuable for detecting the existence of EntP specific antibodies in the sera of theP2-KLH and P3-KLH immunized animals and to detect and quantify the EntP in the supernatant ofproducer strains. The anti-P2-KLH sera cross-reacted with the supernatant of a strain producer ofsakacin 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 producerstrain. Western blotting of EntP with the anti-P2-KLH-generated antibodies suggests that purifiedEntP tends to the formation of aggregates with no antimicrobial activity. Monitoring the purification ofEntP with antipeptide antibodies suggests that while the performance of the evaluated purificationprocedures would be reasonably acceptable in terms of recovery of the antimicrobial activity of thebacteriocin, their yield is far from attractive in terms of recovery of the initial concentration of enterocinP.Keywords: Enterococci; bacteriocins; enterocin P; antipeptide antibodies; ELISA