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.
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 microg ml(-1) 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 0.01 microg ml(-1) and 50% binding inhibition was achieved with 0.1 microg PedA1 ml(-1). Similarly, the limits of detection of PedA1 in MRS medium were found to be 5 microg ml(-1) by protein slot-blotting and 0.01 microg ml(-1) by Western blotting. Most importantly, PH1-KLH-generated polyclonal antibodies detected the presence of PedA1 in the supernatants of the producing strains of Pediococcus acidilactici 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.
Martínez
J M
JM
Departamento de Nutrición y Bromatología III, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
Martínez
M I
MI
Herranz
C
C
Suárez
A
A
Fernández
M F
MF
Cintas
L M
LM
Rodríguez
J M
JM
Hernández
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology*; D001430:Bacteriocins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D001667:Binding, Competitive; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D006241:Haptens / Q000276:immunology; D006433:Hemocyanins / Q000276:immunology; D008969:Molecular Sequence Data; D000070017:Pediocins; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011817:Rabbits; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D000906:Antibodies; D000941:Antigens; D001426:Bacterial Proteins; D001430:Bacteriocins; D006241:Haptens; D000070017:Pediocins; D010446:Peptide Fragments; C074288:pediocin PA-1; C087892:cbnBA protein, Carnobacterium piscicola; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin