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 isometric-headed bacteriophage ul36, a member of the P335 species, was isolated 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 characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within 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) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and beta-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 10 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.