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Journal of Dairy Science Vol. 77 No. 12 3552-3557
© 1994 by American Dairy Science Association ®
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Antibody Sandwich Enzyme-Linked lmmunosorbent Assay

Isabel Gonzalez 1, Rosario Martin 1, Teresa Garcia 1, Paloma Morales 1, Bernabe Sanz 1, and Pablo Hernandez 1

1 Departamento de Nutrición y Bromatología III, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

Polyclonal antibodies raised against live cells of Pseudomonas fluorescens AH-70 have been used in a double antibody sandwich ELISA technique for the detection of P. fluorescens and related psychrotrophic bacteria in refrigerated milk. These antibodies, bound to the wells of a microtiter plate, were used to capture the microorganisms on milk samples. Further immunorecognition of the captured antigens was attained with the same antibodies conjugated to biotin. ExtrAvidin®-peroxidase was used to detect the biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate showed clear differences in absorbance after assay of milk samples containing P. fluorescens strains of different origin and related psychrotrophic microorganisms. The detection threshold for the sandwich ELISA assay developed in this work was 105 cfu/ml.

Key Words: refrigeration of milk • Pseudomonas fluorescens • psychrotrophic bacteria • enzyme-linked immunosorbent assay

Submitted on March 14, 1994
Accepted on July 11, 1994






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Copyright © 1994 by the American Dairy Science Association ®.