Monoclonal antibodies raised against native major capsid proteins of lactococcal c2-like bacteriophages

Abstract

Phage Q38, a representative member of the c2 species, was purified by CsCl gradient and used to immunize BALB/c mice. Monoclonal antibodies (MAbs) were raised and then characterized by enzyme-linked immunosorbent assay. Two MAbs of isotype immunoglobulin G2a, designated 2A5 and 6G7, reacted only with phages belonging to the c2 species and not with phages of the 936 and P335 species, with a Lactococcus lactis cell extract, or with phage DNA. Immunoelectron microscopy showed that both MAbs recognized only phage head proteins. They did not react with any denatured phage proteins in Western blot assays. However, when the nitrocellulose membranes were treated with a Triton-based buffer to assist in protein renaturation, MAbs 2A5 and 6G7 recognized the two major capsid proteins with molecular masses of 80 and 170 kDa. Competitive inhibition tests showed that the two MAbs bind to overlapping epitopes. These MAbs may be a useful tool for monitoring c2 bacteriophages during dairy fermentation and in genetic studies.

FIG. 1

Restriction patterns (EcoRI) of phage DNA (A) and corresponding Southern blot probed with phage Q38 DNA (B). Lanes 1 and 7, 1-kb DNA ladder (1.0, 1.6, 2.1, 3.1, 4.1, 5.1, 6.1, 7.1, 8.1, 9.1, 10.2, 11.2, and 12.2 kb) (GIBCO/BRL); lanes 2, phage eb1; lanes 3, phage c21; lanes 4, phage ml3; lanes 5, phage Q38; lanes 6, phage Q44; lanes 8, phage p2 (936 species); lanes 9, phage ul36 (P335 species).

FIG. 2

Immunoelectron microscopy with gold-labeled protein A (particle size, 10 nm). (A) Phage Q38; (B) MAb 6G7; (C) MAb 2A5.

FIG. 3

Profiles of phage structural proteins as determined by SDS–15% PAGE with the corresponding immunoblots for MAbs 2A5 and 6G7. Phage proteins were stained with Coomassie blue. Lane 1, phage Q44; lane 2, phage Q38; lane 3, phage c2; lane 4, prestained molecular mass markers (25, 32.5, 47.5, 62, 83, and 175 kDa) (New England Biolabs); lane 5, MAb 2A5 and phage c2; lane 6, MAb 6G7 and phage c2.

FIG. 4

Results of dot immunoassays showing sensitivities of MAbs 2A5 (A) and 6G7 (B).