Immunoblotting to demonstrate antigenic and immunogenic differences among nine standard strains of Clostridium difficile

Abstract

The epidemiology of Clostridium difficile-associated disease is being elucidated with the development of typing schemes for the organism. We recently described a new typing scheme based on the incorporation of [35S]methionine into bacterial proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Nine standard strains were identified. We report here some observations on the antigenic differences among these nine strains when studied by immunoblotting. Type-specific rabbit antiserum was raised against each of the nine standard strains. Immunoblotting of the strains with these antisera demonstrated, in addition to the presence of shared, common proteins, a type-specific response with homologous antisera. When [35S]methionine-labeled C. difficile proteins were immunoblotted with homologous and heterologous antisera, both the immunoblots and the autoradiographs demonstrated the same strainspecific response. These strain-specific proteins, which have been so useful for epidemiological and typing purposes, were also immunogenic. We previously reported a typing scheme for Clostridium

Variation in the surface layer proteins of Clostridium difficile

“…Further analysis using immunoblotting can distinguish strains with similar protein pro¢les as there appears to be little antigenic cross-reactivity between strains unless they share identical S-layer proteins. This agrees with earlier work which stated that any single strain only reacts with homologous antiserum [13,19].…”

Immunoblotting and dot blotting

“…The epitopes of various proteins have been mapped and, in some cases, correlated with bio- Luzio and Jackson (1988) Jarausch and Kadenbach (1985) Vartio et al (1982); Dziadek et al (1983) Cohen et al (1986) Costa et al (1986) Mendel-Hartvig and Nelson (1983) Sheng et al (1988) Samson (1986) Yurchenco et al (1982 Reiser and Wardale (1981) Delia et al (1987) Dunn et al ( ) King et al (1985; Cevenini et al (1986); Caldwell et al (1987); Patel et al (1988); Heard et al (1986); Rautenberg et al (1986) Swanson andBarrera (1983); Hadfield and Glynn (1984); Schalla et al (1986) Chapman et al (1987; Kelson et al (1988) De Jongh Leuvenink et al (1985; Sturm et al (1984) Coil et al (1986); Wedege and Froholm (1986); Wedege et al (1988) Aznar et al (1988) Andersen et al (1986; Coates et al (1986);Milner et al (1987); Van Vooren et al (1988) Kermy and Cartwright (1984); Kibe et al (1985); Jacobs et al (1986); Andersen et al (1987); Sasaki et al (1987); Young and Ross ( Battaglia et al…”

Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile

“…Nine standard strains of C. dlfficile, designated A-E and W-Z, were used [5,6] . The identity of the clostridal strains was confirmed by smell, colony morphology and gas-liquid chromatographic analysis of volatile fatty acids [7].…”

“…Three types of antigen were prepared for rabbit immunisations: i) C difficile whole cells from the 9 standard typed strains (A E and W-Z, identified by 35S-methionine labelled protein profiles [6]), were prepared from formalised cell antigens as described previously [5]. ii) E. coli lysates containing cloned C. difficile antigens were prepared from a confluent lawn of plaques on 10 plates (90 mm diameter) overlaid with 0.5 ml SM buffer (0.1 M NaC1, 10 mM MgSO4, 50 mM Tris (ph 7.5), 0.01% gelatine) for 1 h at 25 ° C. The plate lysate suspension was centrifuged (Hi-Spin 21 MSE Scientific Instruments, U.K.) at 7000 × g for 15 rain and the supernatant was used for rabbit immunisation.…”

“…We have previously reported the presence of possible common antigens from C. difficile [5]. In this paper we report the cloning and expression in Escherichia coli of a 70 kDa surface antigen common to the C. difficile species, which may have potential diagnostic applications.…”