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The development and duration of neutralizing antibodies (antitoxin) to pertussis toxin were studied in 38 patients with culture-verified infections due to Bordetella pertussis and one patient with infection due to Bordetella parapertussis. An in vitro neutralization test in microplate culture of Chinese hamster ovary cells was used. An antitoxin response was recorded in 36 patients, the exceptions being two patients treated early with erythromycin (one of whom developed clinical pertussis two years later) and the patient with infection due to B. parapertussis. A long-term follow-up for several months to several years after disease showed maintenance of high antitoxin levels. These results are in accordance with the hypothesis that antibodies to pertussis toxin mediate long-term immunity to whooping cough.

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...

Dermonecrotic toxin (DNT), lipopolysaccharide (LPS), and lymphocytosispromoting factor (LPF) were isolated from Bordetella pertussis and tested for neuroactivity. When injected intraperitoneally into rats, a dose of 0.13 mg of LPF per kg elevated the cyclic GMP level in cerebellum by approximately 70%, whereas DNT (0.5 mg/kg) and LPS (1.5 mg/kg) were without effect. This action of

Chronic pertussis toxin treatment (5 days) of NG108-15 neuroblastoma X glioma hybrid cells had no significant effect on basal cyclic AMP levels whereas it effectively blocked the inhibitory action of acute (10 min) exposure of carbachol (10(-4)M) on intracellular cyclic AMP accumulation, stimulated by prostaglandin E1. This action of pertussis toxin was found to be long lasting: exposure of the cells to pertussis toxin (100 ng/ml) for only 24 h followed by a 5-day withdrawal period still was shown effective on day 7 in abolishing the inhibitory action of carbachol on prostaglandin E1-stimulated cyclic AMP production. Chronic exposure (5 days) of NG108-15 cells to carbachol (10(-5)M) causes an increase in basal cyclic AMP levels by 98%, and a desensitization of the muscarinic inhibition of cyclic AMP accumulation, assessed after a 24-h withdrawal period. When carbachol treatment is carried out in the presence of pertussis toxin (100 ng/ml) both of these effects of carbachol are abolished.