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The application of immunology to food science, two studies : production of monoclonal antibodies (Mabs) specific for an enteropathogenic E. coli (EPEC) ; development of an enzyme-linked immunosorbent assay (ELISA) for [Beta]-N-acetylglucosaminidase (NAGase)

University of British Columbia

Two hybridoma clones, labelled 4D10 C1 and 2H4 H12, produced monoclonal antibodies which recognized the outer membrane of an enteropathogenic Escherichia coli (EPEC) 0142:K86:H6 in an enzyme-linked immunosorbent assay (ELISA) and the whole cell in an immunofluorescence assay. Large scale production of the monoclonal antibodies was accomplished through ascites production in balb/c mice. Purification of the ascites fluid was achieved by gel filtration and ion exchange chromatography. Isotyping of the purified fractions showed 4D10 C1 to be an IgG2 and 2H4 H12 an IgM. These monoclonal antibodies were screened by immunofluorescence assay against several pathogenic and nonpathogenic strains of E.coli in addition to other Enterobacteriaciae. Results of the screening showed these antibodies to be specific for the E.coli serotype to which they were raised. Minimal cross-reactivity with other Enterobacteriaceae was observed. In a separate and concurrent project, the use of an ELISA capable of detecting ß-N-acetylglucosaminidase (NAGase) was examined. White Leghorn hens were injected with commercially prepared bovine NAGase. Eggs were collected and the immunoglobulin fraction separated from the egg yolk by polyethylene glycol precipitation followed by ion exchange on a DEAE-Sephacel column. The use of the purified immunoglobulins was examined in a sandwich, double-sandwich and a competitive ELISA. A statistically significant standard curve for the detection of NAGase was successfully derived using a double-sandwich ELISA when rabbit immunoglobulin was used to coat the microwell plates. This assay was used to measure the NAGase concentration in press juice and fish extract of fresh and frozen salmon muscle samples. The ratio of the NAGase concentration in the press juice to the total NAGase concentration was compared. No significant difference was found between the calculated concentration ratios of the fresh muscle samples and samples frozen for 1 week at -20°C.

Text

2010-10-05

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http://hdl.handle.net/2429/28947

Food Science

University of British Columbia

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