Heron 1. The interaction between pertussis toxin and 10 monoclonal antibodies. Acta path. microbiol. immunol. scand. Sect. c, 95: [177][178][179][180][181][182][183][184][185][186][187] 1987.Data on the epitope specificity of 10 monoclonal hybridoma antibodies (Mabs) that showed positive reaction in enzyme-linked immunosorbent assay (ELISA) towards pertussins toxin (Ptx) are presented. The relative functional affinity of the Mabs was determined in a catching ELISA system. The Mabs were tested for their ability to inhibit the biological activities of this toxin in two in vifro systems, viz. haemagglutination (HA) and Chinese Hamster Ovary cell (CHO) test, and in three in vivo assays: histamine sensitization (HS). leucocytosis-promoting activity (LP) and protection against intra-cerebral challenge (i.c.) with virulent B. pcv-rrrs.ris organisms. Four Mabs were found inhibiting HA and three inhibited the effect on CHO cells. Two Mabs showed demonstrable protective effect on mice in i.c. test. The same two Mabs were also able to inhibit HS and LP activity of Ptx. Five of the ten Mabs reacted with Ptx subjected to blotting after separation of the toxin subunits in sodium-dodecyl sulphate polyacrylamide gel electrophoresis. The five Mabs all bound to more than one subunit. The epitopes defined by several of the Mabs might be useful in the context of a third-generation whooping cough vaccine.Vaccine The vaccine used was the conventional Danish pertussis vaccine consisting of a mixture of killed cells from four local B. pertussis production strains. The bacteria were grown on agar plates in Bordet-Gengou medium (BG) and inactivated with formaldehyde (0.04 v/v %). One ml of vaccine contains 16 x loy cells. The vaccine contains no adjuvant. Ptx content as measured by leucocytosis promoting activity test is 15 LP units/ml of vaccine.Bacterial extracts. One of the B. pertussis production strains (strain no. 3843) was cultured on BG-medium for 3 days. The colonies from one petri dish with a diameter of I5 cm were inoculated into 200 ml Stainer-Scholte medium (24) and grown as shaker-flask cultures for 24 or 48 hours.Cultures were harvested by centrifugation. Supernatants were kept at 4 "C as pertussis culture supernatants.The pellet was resuspended in 5 ml saline (9 g NaCI/I + 100 mg Thiomersal/l) and sonicated for 3 x 45 s. The preparation was centrifuged and the supernatant kept at 4 "C as pertussis sonicate; the pellet was discharded.Sonicate and supernatant contained about 10 LP units/ ml. Pertiissis toxin (Ptx]. Ptx purified from B. pertussis strain 3779 was a kind gift from Dr. John J. Munoz ( I , 13. 14). The Ptx was supplied in crystalline form and dissolved to a final concentration of 0.2 mg/ml in equal volumes of FAAB-buffer (0.08 DL-alpha-alanin, 0.05 M formic acid, pH 3.5) and TNB-buffer (0.05 M Tris. I M NaCI. pH 8.0).Analyzed in SDS-PAGE under non-reducing conditions. Ptx was found to comprise 5 polypeptide chains of molecular weight 28,000 (SI), 26,500 (S2). 25.000 (S3) 13.500 (S4) and 12,500 (S5).Protein quanti...
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