Personne : Pandian, Sithian
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Pandian
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Sithian
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Université Laval. Département de sciences et technologie des aliments
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ncf12004008
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Publication Restreint 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(American Society for Microbiology, 1993-07-01) Hébert, Jacques; Moineau, Sylvain; Jobin, Marie; Pandian, Sithian; Klaenhammer, Todd R.; Bernier, DenisThe 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 β-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 107 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.Publication Restreint Differentiation of two abortive mechanisms by using monoclonal antibodies directed toward Lactococcal bacteriophage capsid proteins(American Society for Microbiology, 1993-01-01) Moineau, Sylvain; Durmaz, Evelyn; Pandian, Sithian; Klaenhammer, Todd R.Monoclonal antibodies were used to monitor the accumulation of the major capsid protein of the lactococcal small isometric bacteriophage u136 (P335 species) over the course of a one-step growth curve. A sandwich enzyme-linked immunosorbent assay was then used to distinguish two abortive phage resistance mechanisms, Hsp and Prf. Capsid protein production of u136 was almost totally inhibited by the Hsp-induced abortive mechanism, supporting previous data that this mechanism blocks phage DNA replication. Prf-induced abortive infection only partially (50%) inhibited capsid protein production, suggesting that this mechanism targets some other point, perhaps within transcription or translation processes. The results confirmed that Hsp and Prf act at different targets in the phage lytic cycle. Use of monoclonal antibodies also demonstrated that production of the major capsid protein is a nonlimiting step in the lytic cycle of lactococcal phage u136.