Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.
Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.
Suárez
A M
AM
Departmento de Nutrición y Bromatologia III, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.
Rodríguez
J M
JM
Hernández
P E
PE
Azcona-Olivera
J I
JI
D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000276:immunology* / Q000592:standards; D000915:Antibody Affinity; D000917:Antibody Formation; D000918:Antibody Specificity; D002772:Cholera Toxin / Q000276:immunology; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005260:Female; D006433:Hemocyanins / Q000276:immunology; D007114:Immunization / Q000379:methods; D018796:Immunoconjugates / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009561:Nisin / Q000032:analysis* / Q000276:immunology* / Q000592:standards; D012015:Reference Standards
D000900:Anti-Bacterial Agents; D018796:Immunoconjugates; D009561:Nisin; D002772:Cholera Toxin; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin