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Applied and Environmental Microbiology, November 1998, p. 4536-4545, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Generation of Polyclonal Antibodies of
Predetermined Specificity against Pediocin PA-1
José M.
Martínez,
María I.
Martínez,
Ana M.
Suárez,
Carmen
Herranz,
Pilar
Casaus,
Luis M.
Cintas,
Juan M.
Rodríguez, and
Pablo E.
Hernández*
Departamento de Nutrición y
Bromatología III, Facultad de Veterinaria, Universidad
Complutense, 28040 Madrid, Spain
Received 21 January 1998/Accepted 13 August 1998
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.
*
Corresponding author. Mailing address: Departamento de
Nutrición y Bromatología III, Facultad de Veterinaria,
Universidad Complutense, 28040 Madrid, Spain. Phone: 34-91-3943752. Fax: 34-91-3943743. E-mail:
ehernan{at}eucmax.sim.ucm.es.
Applied and Environmental Microbiology, November 1998, p. 4536-4545, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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