Purification and Characterisation of a Fimbrial Haemagglutinin from Bordetellapertussis for Use in an Enzyme-linked Immunosorbent Assay

Abstract

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...

Galanin receptor and its ligands in the rat hippocampus

“…Pertussis toxin was prepared as described in [IS] and generously supplied by Dr Askelof, Stockholm. The antisera against pertussis toxin was raised in rabbits and tested for ability to inhibit the morphological effects of pertussis toxin on Chinese hamster ovary cells [15]. All the other reagents used were of analytical grade.…”

“…The first incubation was carried out for I h at 37"C, followed by the second addition and incubation for 15 these peptides, except the different kinds of porcine and rat galanin, gave a significant inhibition of '2sI-galanin binding. The classical neurotransmitters, 5-hydroxytryptamine, y-aminobutyric acid and noradrenaline (100 pM) did not influence the specific binding of 1 .O nM 12'1-galanin, measured in ventral hippocampal membranes under equilibrium conditions.…”

Detection of Antibodies in Human Serum Against the Fimbrial Haemagglutinin of Bordetella Pertussis by Enzyme-linked Immunosorbent Assay

“…An enzyme-substrate reaction time of 1 h at 37°C was used throughout the study. More details of the test are given in the previous paper (Askelof et al, 1982).…”

“…In particular, the risk of the more uncommon but severe neurological complications has been emphasised. The protective efficacy of some vaccines has also been questioned, because high attack rates have been reported in fully vaccinated children (Stewart, 1977;Banck, Broms and Ljung, 1980). These considerations have led to low acceptance rates for the vaccine in Great Britain (Pollard, 1980) and a withdrawal of pertussis vaccine in Sweden.…”

Partial characterisation of a soluble haemagglutinin from human diarrhoeal isolates of Aeromonas

“…Previous studies of the fimbrial and heat-labile agglutinins of Bordetella pertussis (Askelof et al, 1982) indicated that a high salt concentration (0.5 M NaCl) inhibited haemagglutination by fimbrial but not by heat labile haemagglutinin. Thus, haemagglutination experiments with gel-filtration fractions from the strain of A .…”