Vol. 28, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1990, p. 182-187 0095-1137/90/020182-06$02.00/0 Copyright © 1990, American Society for Microbiology
Serological Typing of Branhamella catarrhalis Strains on the Basis of Lipopolysaccharide Antigens MARIO VANEECHOUTTE,* GERDA VERSCHRAEGEN, GEERT CLAEYS, AND ANNE-MARIE VAN DEN ABEELE Department of Medical Microbiology, University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium Received 12 April 1989/Accepted 12 October 1989 A total of 302 strains of Branhamella catarrhalis from different parts of the world were serologically typed according to their lipopolysaccharide (LPS) antigenicity. For this purpose, an inhibition enzyme-linked immunosorbent assay was developed using the following reagents: antisera raised against whole bacterial suspensions for a panel of 16 serotype strains and LPS prepared from these strains by phenol extraction. Antisera were absorbed with whole bacterial suspensions of the B. catarrhalis strains to be tested. The residual activity of the sera against the homologous LPS was determined by means of an enzyme-linked immunosorbent assay, using microdilution plates coated with LPS. Strains which gave >90% reduction of activity were considered to carry the same LPS type as the serotype strain. It was shown that 93.4% of the strains tested carried one of three possible LPS types. LPS of B. catarrhalis are the rough type and have an apparent Mr of 5,500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Antisera. Antisera to 16 randomly chosen strains of B. catarrhalis were produced by subcutaneous injection of rabbits on days 0, 21, and 42 with 500-,ul aliquots of saline suspensions containing 108 CFU of living bacteria per ml. Rabbits were exsanguinated on day 52. Antiserum titers were determined by using an ELISA, with whole bacteria as antigen, essentially by the method of Voller et al. (26). Typing antigens. From the 16 serotype strains used to raise rabbit antisera, different typing antigens were prepared and used to coat the ELISA microdilution plates. (i) NaOH extracts of whole bacteria. The serotype strains were grown overnight at 37°C in tryptone soya broth (Oxoid Ltd., Basingstoke, United Kingdom) on a rotary shaker. Cells were precipitated by centrifugation and washed once in saline. One gram of wet cells was suspended in 15 ml of 0.4 N NaOH and kept at 50°C for 1 h. The pH was readjusted to 9.6, and the suspension was centrifuged for 20 min at 3,000 x g. The supernatant was used as the coating antigen. (ii) Protease-treated bacteria. Whole bacteria were washed and treated with 100 ,ug of protease XIV (Sigma Chemical Co., St. Louis, Mo.) per mg of protein for 4 h at 37°C. Protease activity was stopped by boiling for 5 min. After centrifugation for 30 min at 3,000 x g, the supernatant was used to coat ELISA microdilution plates. (iii) Phenol-water-extracted LPS. A 3-g (wet weight) portion of bacteria was obtained from each of the 16 serotype strains from the overnight growth of 10 human blood agar plates. This suspension was washed once in saline and lyophilized, yielding 0.5 g of dry weight. The resulting powder was extracted with phenol water, using the method of Westphal and Jann (27). The lipopolysaccharides (LPS) were further purified of DNA contamination by repeated ultracentrifugation until the optical density (OD) of the supernatant, measured at 280 nm, was 90%. In most cases, the inhibition was >95%. Moreover, in most ELISAs a clear gap between homologous and heterologous inhibition was observed. The strains which belong to a type other than that of the homologous serotype strain, as assessed in previous tests, gave inhibition percentages scattered between 0 and 80% (Fig. 2). For example, strain 11i, which belongs to the same type as serotype strains J9 and F17 (Fig. 2c and d, >90% inhibition) gave inhibition percentages of
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1990, p. 182-187 0095-1137/90/020182-06$02.00/0 Copyright © 1990, American Society for Microbiology
Serological Typing of Branhamella catarrhalis Strains on the Basis of Lipopolysaccharide Antigens MARIO VANEECHOUTTE,* GERDA VERSCHRAEGEN, GEERT CLAEYS, AND ANNE-MARIE VAN DEN ABEELE Department of Medical Microbiology, University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium Received 12 April 1989/Accepted 12 October 1989 A total of 302 strains of Branhamella catarrhalis from different parts of the world were serologically typed according to their lipopolysaccharide (LPS) antigenicity. For this purpose, an inhibition enzyme-linked immunosorbent assay was developed using the following reagents: antisera raised against whole bacterial suspensions for a panel of 16 serotype strains and LPS prepared from these strains by phenol extraction. Antisera were absorbed with whole bacterial suspensions of the B. catarrhalis strains to be tested. The residual activity of the sera against the homologous LPS was determined by means of an enzyme-linked immunosorbent assay, using microdilution plates coated with LPS. Strains which gave >90% reduction of activity were considered to carry the same LPS type as the serotype strain. It was shown that 93.4% of the strains tested carried one of three possible LPS types. LPS of B. catarrhalis are the rough type and have an apparent Mr of 5,500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Antisera. Antisera to 16 randomly chosen strains of B. catarrhalis were produced by subcutaneous injection of rabbits on days 0, 21, and 42 with 500-,ul aliquots of saline suspensions containing 108 CFU of living bacteria per ml. Rabbits were exsanguinated on day 52. Antiserum titers were determined by using an ELISA, with whole bacteria as antigen, essentially by the method of Voller et al. (26). Typing antigens. From the 16 serotype strains used to raise rabbit antisera, different typing antigens were prepared and used to coat the ELISA microdilution plates. (i) NaOH extracts of whole bacteria. The serotype strains were grown overnight at 37°C in tryptone soya broth (Oxoid Ltd., Basingstoke, United Kingdom) on a rotary shaker. Cells were precipitated by centrifugation and washed once in saline. One gram of wet cells was suspended in 15 ml of 0.4 N NaOH and kept at 50°C for 1 h. The pH was readjusted to 9.6, and the suspension was centrifuged for 20 min at 3,000 x g. The supernatant was used as the coating antigen. (ii) Protease-treated bacteria. Whole bacteria were washed and treated with 100 ,ug of protease XIV (Sigma Chemical Co., St. Louis, Mo.) per mg of protein for 4 h at 37°C. Protease activity was stopped by boiling for 5 min. After centrifugation for 30 min at 3,000 x g, the supernatant was used to coat ELISA microdilution plates. (iii) Phenol-water-extracted LPS. A 3-g (wet weight) portion of bacteria was obtained from each of the 16 serotype strains from the overnight growth of 10 human blood agar plates. This suspension was washed once in saline and lyophilized, yielding 0.5 g of dry weight. The resulting powder was extracted with phenol water, using the method of Westphal and Jann (27). The lipopolysaccharides (LPS) were further purified of DNA contamination by repeated ultracentrifugation until the optical density (OD) of the supernatant, measured at 280 nm, was 90%. In most cases, the inhibition was >95%. Moreover, in most ELISAs a clear gap between homologous and heterologous inhibition was observed. The strains which belong to a type other than that of the homologous serotype strain, as assessed in previous tests, gave inhibition percentages scattered between 0 and 80% (Fig. 2). For example, strain 11i, which belongs to the same type as serotype strains J9 and F17 (Fig. 2c and d, >90% inhibition) gave inhibition percentages of
