title: Development of an antigen-capture enzyme-linked immunosorbent assay for Clostridium perfringens beta2-toxin in porcine feces and the neonatal piglet intestine.
abstract: An enzyme-linked immunosorbent assay (ELISA) was developed for detection and quantitation of beta2-toxin in neonatal piglet intestinal contents. Polystyrene plates were coated with polyclonal capture antibodies prepared against consensus recombinant beta2-toxin. The ELISA was developed using consensus recombinant beta2-toxin, atypical recombinant beta2-toxin, purified consensus native beta2-toxin, and field samples of neonatal porcine intestinal contents. Captured antigen was detected using a horseradish peroxidase-labeled monoclonal antibody against consensus recombinant beta2-toxin. The limit of detection of the ELISA for consensus beta2-toxin was between 2.0 and 3.5 ng/ml. The ELISA detected atypical recombinant beta2-toxin only weakly. Optical density was protein concentration dependent. The test confirmed differences between consensus and atypical recombinant beta2-toxin, but similar results obtained when testing pure consensus recombinant beta2-toxin and native beta2-toxin. Results obtained from intestinal content samples, particularly from the small intestine, were highly inconsistent and suggested variable protease activity. Addition of protease inhibitors partially prevented degradation of the toxin; however, sample processing at low temperature, at a lower pH (citrate buffer with 5% of bovine serum albumin, pH 6.1), and "cold incubation" of applied antigens abolished protease activity. The recombinant toxin was preserved in spiked intestinal samples by freezing at -70°C, suggesting that necropsy samples can be stored frozen for periodic testing. With appropriate sample preparation, antigen-capture ELISA can detect beta2-toxin in the intestinal content and feces of neonatal piglets.
authors.affiliation: Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Kircanski
authors.forename: Douglas
authors.initials: D
authors.lastname: Hodgins
authors.forename: Glenn
authors.initials: G
authors.lastname: Soltes
authors.forename: Yanlong
authors.initials: Y
authors.lastname: Pei
authors.forename: Valeria R
authors.initials: VR
authors.lastname: Parreira
authors.forename: J Glenn
authors.initials: JG
authors.lastname: Songer
authors.forename: John F
authors.initials: JF
authors.lastname: Prescott
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001427:Bacterial Toxins / Q000302:isolation & purification*; D003015:Clostridium Infections / Q000175:diagnosis / Q000662:veterinary*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005243:Feces / Q000737:chemistry*; D005615:Freezing; D007422:Intestines / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis*
chemical_list: D001427:Bacterial Toxins; D011994:Recombinant Proteins; C109896:cpb2 protein, Clostridium perfringens