PRODUCTION OF MONOCLONAL-ANTIBODIES AGAINST THE MAJOR CAPSID PROTEIN OF THE LACTOCOCCUS BACTERIOPHAGE-UL36 AND DEVELOPMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR DIRECT PHAGE DETECTION IN WHEY AND MILK

The only major structural protein (35 kDa) of the lactococcal small is ometric-headed bacteriophage ul36, a member of the P335 species, was i solated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterize d. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage stru cture. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Im munoelectron microscopy showed that these two proteins are localized w ithin the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) wa s developed for direct detection of lactococcal phages in whey and mil k samples. Whey and milk components, however, interfered with the cond uct of the assay. Partial denaturation of milk samples by heat treatme nt in the presence of SDS and beta-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With t he method used here, 10(7) PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.