Serotyping of Listeria monocytogenes by enzyme-linked immunosorbent assay and identification of mixed-serotype cultures by colony immunoblotting

Abstract

Routine analysis of Listeria monocytogenes by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination. We have adapted a commercially available set of L. monocytogenes antisera to an enzyme-linked immunosorbent assay (ELISA) format for high-throughput, low-cost serotype determination. Rather than subjective visualization of agglutination, positive antigen and antiserum reactions were scored by a quantitative, colorimetric reaction. ELISA serotyping of 89 of 101 L. monocytogenes isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously by the ELISA method. In addition, mixed-serotype cultures of L. monocytogenes were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining.

FIG. 1.

ELISA O-antigen and H-antigen reactions of serogroup 1/2 and 3 L. monocytogenes strains. (A) Strain J0098, serotype 1/2a; (B) strain G848, serotype 1/2b; (C) strain G-3321, serotype 1/2c; (D) strain J0095, serotype 3a; (E) strain cpp81, serotype 3b; (F) strain J0096, serotype 3c.

FIG. 2.

ELISA O-antigen reactions of serogroup 4 L. monocytogenes strains. (A) Strain FSL-X1-010, serotype 4a; (B) strain JL2-10, serotype 4ab; (C) strain J0097, serotype 4b; (D) strain J0099, serotype 4c; (E) strain J0094, serotype 4d; (F) strain 19118, serotype 4e. Note that serotype 4e is indistinguishable from 4b in this case.

FIG. 3.

Colony immunoblotting of single- and mixed-serotype cultures of L. monocytogenes. Blots of serotype 1/2a strain J0098 (A), serotype 4b strain J0097 (B), or a mixture of strains J0098 and J0097 (C) were probed sequentially with O-factor antiserum I/II (stained red) and O-factor antiserum V/VI (stained green). (D) A streak plate of strain 19118 was blotted and probed sequentially with O-factor antiserum V/VI (stained red) and O-factor antiserum I/II (stained green). Insets are magnifications (×5) of the indicated region of each filter to show detail.