1887

Microbiology Society logo

Volume 145, Issue 10

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 Free

Abstract

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 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 001 μg ml and 50% binding inhibition was achieved with 01 μg PedA1 ml. Similarly, the limits of detection of PedA1 in MRS medium were found to be 5 μg ml by protein slot-blotting and 001 μg ml by Western blotting. Most importantly, PH1–KLH-generated polyclonal antibodies detected the presence of PedA1 in the supernatants of the producing strains of 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.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): ADT, agar diffusion test , bacteriocin , CB, coating buffer , CD-, CI-, NCI-and S-ELISA, competitive direct, competitive indirect, non-competitive indirect and sandwich ELISA , immunodetection , KLH, keyhole limpet haemocyanin , LAB, lactic acid bacteria , lactic acid bacteria , MPA, microtitre plate assay , OA, ovalbumin , PedA1, pediocin PA-1 , pediocin PA-1 and TMB, 3,3′,5,5′-tetramethylbenzidine
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-10-2777
1999-10-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/10/1452777a.html?itemId=/content/journal/micro/10.1099/00221287-145-10-2777&mimeType=html&fmt=ahah

References

  1. Abriouel, H., Valdivia, E., Gálvez, A. & Maqueda, M. (1998). Response of Salmonella cholerasuis LT2 spheroplasts and permeabilized cells to the bacteriocin AS-48. Appl Environ Microbiol 64, 4623-4626. [Google Scholar]
  2. Avrameas, S. & Ternynck, T. (1969). The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6, 53-56.[CrossRef] [Google Scholar]
  3. Aymerich, T., Holo, H., Havarstein, L. S., Garriga, M. & Nes, I. F. (1996). Biochemical and genetic characterization of enterocin A from Enterococcusfaecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl Environ Microbiol 62, 1676-1682. [Google Scholar]
  4. van Belkum, M. J., Worobo, R. W. & Stiles, M. (1997). Double-glycine-type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis. Mol Microbiol 23, 1293-1301.[CrossRef] [Google Scholar]
  5. Bennik, M. H. J., Smid, E. J. & Gorris, L. G. M. (1997). Vegetable-associated Pediococcus parvulus produces pediocin PA-1. Appl Environ Microbiol 63, 2074-2076. [Google Scholar]
  6. Bennik, M. H. J., Vanloo, B., Brasseur, R., Gorris, L. G. M. & Smid, E. J. (1998). A novel bacteriocin with a YGNGV motif from vegetable-associated Enterococcus mundtii: full characterization and interaction with target organisms. Biochim Biophys Acta 1373, 47-58.[CrossRef] [Google Scholar]
  7. Bhugaloo-Vial, P., Dousset, X., Metivier, A., Sorokine, O., Anglade, P., Bogaval, P. & Marion, D. (1996). Purification and amino acid sequences of piscicocins V1a and V1b, two class IIa bacteriocins secreted by Carnobacterium piscicola V1 that display significant levels of specific inhibitory activity. Appl Environ Microbiol 62, 4410-4416. [Google Scholar]
  8. Bhunia, A. K. (1994). Monoclonal antibody-based enzyme immunoassay for pediocins of Pediococcus acidilactici. Appl Environ Microbiol 60, 2692-2696. [Google Scholar]
  9. Bhunia, A. K., Johnson, M. C. & Ray, B. (1987). Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Ind Microbiol 2, 319-322.[CrossRef] [Google Scholar]
  10. Bhunia, A. K., Johnson, M. C., Ray, B. & Elden, E. L. (1990). Antigenic property of pediocin AcH produced by Pediococcus acidilactici H. J Appl Bacteriol 69, 211-215.[CrossRef] [Google Scholar]
  11. Biet, F., Berjeaud, J. M., Worobo, R. W., Cenatiempo, Y. & Fremaux, C. (1998). Heterologous expression of the bacteriocin mesentericin Y105 using the dedicated transport system and the general secretion pathway. Microbiology 144, 2845-2854.[CrossRef] [Google Scholar]
  12. Bouksaim, M., Fliss, I., Meghrous, J., Simard, R. & Lacroix, C. (1998). Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence. J Appl Microbiol 81, 176-184. [Google Scholar]
  13. Briand, J. P., Muller, S. & van Regenmortel, M. H. V. (1985). Synthetic peptides as antigens: pitfalls of conjugation methods. J Immunol Methods 78, 59-69.[CrossRef] [Google Scholar]
  14. Casaus, P., Nilsen, T., Cintas, L. M., Nes, I. F., Hernández, P. E. & Holo, H. (1997). Enterocin B, a new bacteriocin from Enterococcusfaecium T136 which can act synergistically with enterocin A. Microbiology 143, 2287-2294.[CrossRef] [Google Scholar]
  15. Cintas, L. M., Casaus, P., Havarstein, L. S., Hernández, P. E. & Nes, I. F. (1997). Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl Environ Microbiol 63, 4321-4330. [Google Scholar]
  16. Cintas, L. M., Casaus, P., Holo, H., Hernández, P. E., Nes, I. F. & Havarstein, L. S. (1998). Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to staphylococcal hemolysins. J Bacteriol 180, 1988-1994. [Google Scholar]
  17. Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395.[CrossRef] [Google Scholar]
  18. Ennahar, S., Aoude-Werner, D., Sorokine, O., van Dorsselaer, A., Bringel, F., Hubert, J.-C. & Hasselmann, C. (1996). Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese. Appl Environ Microbiol 62, 4381-4387. [Google Scholar]
  19. Falahaee, M. B., Adams, M. R., Dale, J. W. & Morris, B. A. (1990). An enzyme immunoassay for nisin. Int J Food Sci Technol 25, 590-595. [Google Scholar]
  20. Gasson, M. J. (1983). Plasmid complements of Streptococcus lactis NCDO712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154, 1-9. [Google Scholar]
  21. Groome, N. P. (1994). Immunoassays of proteins and anti-peptide antibodies. In Peptide Antigens: A Practical Approach, pp. 139-179. Edited by G. R. Wisdom. Oxford: IRL Press.
  22. Henderson, J. T., Chopko, A. L. & Wassenaar, P. D. (1992). Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC1.0. Arch Biochem Biophys 295, 5-12.[CrossRef] [Google Scholar]
  23. Holck, A., Axelsson, L., Birkeland, S. E., Aukrust, T. & Blom, H. (1992). Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillussake Lb706. J Gen Microbiol 138, 2715-2720.[CrossRef] [Google Scholar]
  24. Horn, N., Martı́nez, M. I., Martı́nez, J. M., Hernández, P. E., Gasson, M. J., Rodrı́guez, J. M. & Dodd, H. (1998). Production of pediocin PA-1 by Lactococcus lactis using the lactococcin A secretory apparatus. Appl Environ Microbiol 64, 818-823. [Google Scholar]
  25. Jack, R. W., Tagg, J. R. & Ray, B. (1995). Bacteriocins of Gram-positive bacteria. Appl Environ Microbiol 59, 171-200. [Google Scholar]
  26. Klaenhammer, T. R. (1993). Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev 12, 39-86.[CrossRef] [Google Scholar]
  27. Leistner, L. & Gorris, L. G. M. (1995). Food preservation by hurdle technology. Trends Food Sci Technol 6, 41-46.[CrossRef] [Google Scholar]
  28. Martı́nez, M. I., Rodrı́guez, J. M., Suárez, A., Martı́nez, J. M., Azcona, J. I. & Hernández, P. E. (1997). Generation of polyclonal antibodies against a chemically synthesized N-terminal fragment of the bacteriocin pediocin PA-1. Lett Appl Microbiol 24, 488-492.[CrossRef] [Google Scholar]
  29. Martı́nez, J. M., Martı́nez, M. I., Suárez, A. M., Herranz, C., Casaus, P., Cintas, L. M., Rodrı́guez, J. M. & Hernández, P. E. (1998). Generation of polyclonal antibodies of predetermined specificity against pediocin PA-1. Appl Environ Microbiol 64, 4536-4545. [Google Scholar]
  30. Marugg, J. D., Gonzalez, C. F., Kunka, B. S., Ledeboer, A. M., Pucci, M. J., Toonen, M. Y., Walker, S. A., Zoetmulder, L. C. M. & Vanderbergh, P. A. (1992). Cloning, expression and nucleotide sequence of genes involved in production of pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0. Appl Environ Microbiol 58, 2360-2367. [Google Scholar]
  31. Metivier, A., Pilet, M. F., Dousset, X., Sorokine, O., Anglade, P., Zagorec, M., Piard, J. C., Marion, D., Cenatiempo, Y. & Fremaux, C. (1998). Divercin V41, a new bacteriocin with two disulphide bonds produced by Carnobacterium divergens V41: primary structure and organization. Microbiology 144, 2837-2844.[CrossRef] [Google Scholar]
  32. Nakane, P. K. & Kawoi, A. (1974). Peroxidase-labelled antibody: a new method of conjugation. J Histochem Cytochem 22, 1084-1091.[CrossRef] [Google Scholar]
  33. Nes, I. F., Diep, D. B., Håvarstein, L. S., Brueberg, M. B., Eijsink, V. & Holo, H. (1996). Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Leeuwenhoek 70, 113-128.[CrossRef] [Google Scholar]
  34. Nieto Lozano, J. C., Nissen Meyer, J., Sletten, K., Pelaez, C. & Nes, I. F. (1992). Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici. J Gen Microbiol 138, 1985-1990.[CrossRef] [Google Scholar]
  35. Nissen-Meyer, J. & Nes, I. F. (1997). Ribosomally synthesized antimicrobial peptides: their function, biosynthesis and mechanism of action. Arch Microbiol 167, 67-77.[CrossRef] [Google Scholar]
  36. Piva, A. & Headon, D. H. (1994). Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61. Microbiology 140, 697-702.[CrossRef] [Google Scholar]
  37. Rodrı́guez, J. M., Cintas, L. M., Casaus, P., Suárez, A. & Hernández, P. E. (1995a). PCR detection of the lactocin S structural gene in bacteriocin-producing lactobacilli from meat. Appl Environ Microbiol 61, 2802-2805. [Google Scholar]
  38. Rodrı́guez, J. M., Cintas, L. M., Casaus, P., Horn, N., Dodd, H. M., Hernández, P. E. & Gasson, M. J. (1995b). Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages. J Appl Bacteriol 78, 109-115.[CrossRef] [Google Scholar]
  39. Rodrı́guez, J. M., Cintas, L. M., Martı́nez, M. I., Casaus, P., Suárez, A. M. & Hernández, P. E. (1997). Detection of pediocin PA-1 producing pediococci by rapid molecular biology procedures. Food Microbiol 14, 363-371.[CrossRef] [Google Scholar]
  40. Rolland, M. P., Bitri, L. & Besancon, P. (1995). Monospecificity of the antibodies to bovine αs1-casein fragment 140–149: application to the detection of bovine milk in caprine dairy products. J Dairy Res 62, 83-88.[CrossRef] [Google Scholar]
  41. Shägger, H. & Von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range of 1 to 100 kDa. Anal Biochem 166, 368-379.[CrossRef] [Google Scholar]
  42. Stringer, S. C., Dodd, C. E. R., Morgan, M. R. A. & Waites, W. M. (1995). Locating nisin-producing Lactococcus lactis in a fermented meat system. J Appl Bacteriol 78, 341-348.[CrossRef] [Google Scholar]
  43. Suárez, A. M., Rodrı́guez, J. M., Hernández, P. E. & Azcona-Olivera, J. I. (1996a). Generation of polyclonal antibodies against nisin: immunization strategies and immunoassays development. Appl Environ Microbiol 62, 2117-2121. [Google Scholar]
  44. Suárez, A. M., Rodrı́guez, J. M., Morales, P., Hernández, P. E. & Azcona-Olivera, J. I. (1996b). Development of monoclonal antibodies to the lantibiotic nisin A. J Agric Food Chem 44, 2936-2940.[CrossRef] [Google Scholar]
  45. Suárez, A. M., Azcona, J. I., Rodrı́guez, J. M., Sanz, B. & Hernández, P. E. (1997). One-step purification of nisin A by immunoaffinity chromatography. Appl Environ Microbiol 63, 4990-4992. [Google Scholar]
  46. Tichaczek, P. S., Vogel, R. F. & Hammes, W. P. (1994). Cloning and sequencing of sakP encoding sakacin P, the bacteriocin produced by Lactobacillus sake LTH673. Microbiology 140, 361-370.[CrossRef] [Google Scholar]
  47. Venema, K., Kok, J., Marugg, J. D., Toonen, M. Y., Ledeboer, A. M., Venema, G. & Chikindas, M. L. (1995). Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme. Mol Microbiol 17, 515-522.[CrossRef] [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-10-2777
Loading
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
Microbiology 145, 2777 (1999); https://doi.org/10.1099/00221287-145-10-2777
/content/journal/micro/10.1099/00221287-145-10-2777
/content/journal/micro/10.1099/00221287-145-10-2777
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error