SUMMARY.The fimbrial haemagglutinin (F-HA) of Bordetellapertussis grown on solid medium was extracted with 1 M sodium acetate for 72 h at 20°C, and partially purified by Sephacryl S-300 gel chromatography. A pooled fraction with fimbrial haemagglutinating activity was shown to contain fimbriae of the expected morphology by electron microscopy. Chemical and biological assays showed that the F-HA fraction contained some heat-labile agglutinogen and lipopolysaccharide but no measurable lymphocytosis-promoting factor or heat-labile toxin. The F-HA fraction used as antigen in an enzyme-linked immunosorbent assay (ELISA) permitted the detection of antibodies in convalescent serum from a patient with whooping cough. The impurities, heat-labile agglutinogens and lipopolysaccharide, did not contribute to the ELISA activity. The method for preparation of the F-HA antigen is simple, reproducible and gives a high yield.
INTRODUCTIONThe isolation of Bordetella pertussis from patients with whooping cough is unreliable, especially after the first 2 weeks of infection, and attempts have been made to establish the diagnosis by immunological techniques. Agglutination, complement fixation, haemagglutination inhibition and immunofluorescence have been used to detect antibodies to B. pertussis and either whole bacteria or crude supernates from liquid cultures have been used as antigen (see Granstrom et al., 1982).The use of whole bacteria has made it impossible to develop a specific assay because different antigens, which are not all unique to B. pertussis, are either associated with or released from the bacteria (Pittman, 1979). These include (i) fimbriae, which agglutinate erythrocytes and have been named the fimbrial haemagglutinin (F-HA), (ii) heat-labile agglutinogens, types 1-3, which are believed to be outer-membrane proteins and form the basis for serotyping B. pertussis, (iii) lipopolysaccharide, a structural component of the outer membrane, (iv) heat-labile toxin, a dermonecrotic protein toxin, and (v) the lymphocytosis-promoting factor, identical to the histamine-sensitising factor and the islet-activating protein.We report here the isolation and partial purification of a fimbrial fraction from B. pertussis and its use as an antigen in an enzyme-linked immunosorbent assay (ELISA).
MATERIALS AND METHODSBacterial strains. Bordetella pertussis strains GL353 (serotype l), 360E (serotype 1,2), M2 (serotype 1,3), and 18323 (serotype 1,2,3), B. parapertussis strain ATCC 15237, and B. bronchiseptica strain ATCC 19385 were from the National Bacteriological Laboratory, Stockholm, Sweden. They were held as lyophilised cultures.Bacteria were grown on Bordet-Gengou Agar (Difco Laboratories, Detroit, Mich., USA) containing 30% horse blood. After one subculture of the lyophilised bacteria, 24-h cultures were harvested and suspended in 1~ sodium acetate (Masry, 1952) to a density of about 10I2 bacteria/ml. After extraction for 3 days at room temperature with occasional gentle shaking, the bacteria were centrifuged at 5000 g for 30 min. T...