Generation of polyclonal antibodies of predetermined specificity against pediocin PA-1.
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 microg/ml by immunodotting and 1 microg/ml in the NCI-ELISA. However, the CI-ELISA enhanced the limit of detection of pedA1 to 0.025 microg/ml, while the amount of free pedA1 required for 50% binding inhibition was 10 microg/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 microg/ml, and the 50% binding inhibition value was reduced to 0.5 microg 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.
Martínez
J M
JM
Departamento de Nutrición y Bromatología III, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.
Martínez
M I
MI
Suárez
A M
AM
Herranz
C
C
Casaus
P
P
Cintas
L M
LM
Rodríguez
J M
JM
Hernández
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis / Q000276:immunology; D000906:Antibodies; D000918:Antibody Specificity; D001430:Bacteriocins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006433:Hemocyanins; D008969:Molecular Sequence Data; D000070017:Pediocins; D011817:Rabbits; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000900:Anti-Bacterial Agents; D000906:Antibodies; D001430:Bacteriocins; D000070017:Pediocins; C074288:pediocin PA-1; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin