Date of Award
Doctor of Philosophy
Paul A. Hartman
Coagglutination tests with polyclonal antibodies were developed for the detection of Escherichia coli [beta]-galactosidase (GAL), [beta]-glucuronidase (GUD), and glutamate decarboxylase (GAD) in cell lysates. All three enzymes were detected in 93% of the E. coli tested; two of the three enzymes were detected in the remaining 7%. Among 42 non-E. coli, two (5%) were agglutinated by all three anti-enzyme conjugates, two (5%) were agglutinated by two conjugates, six (14%) were agglutinated by a single conjugate, and 32 (76%) were not agglutinated by any of the conjugates;Enzyme-capture assays with polyclonal antibodies detected the presence of GAL in seven of eight and GUD in all eight E. coli tested. Some GAL-positive Citrobacter freundii and Enterobacter cloacae also were positive by the enzyme-capture assay, indicating that the antibodies were not specific for E. coli GAL. Five other GAL-positive non-E. coli were negative by the enzyme-capture assay;The coagglutination tests and the enzyme-capture assay were rapid methods for the detection of GAL, GUD, and GAD in cell lysates;Polyclonal antibodies to heat-treated E. coli differed in the numbers of E. coli and non-E. coli detected by enzyme immunoassay. Antiserum to one E. coli strain reacted with all of the bacteria tested while antiserum to a second strain was positive for just two of five E. coli tested, Enterobacter agglomerans and Klebsiella ozanae. Attempts to remove the cross-reacting antibodies by adsorption were unsuccessful;Monoclonal antibodies to heat-treated E. coli varied in specificity. Antibodies from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of the 13 non-E. coli tested, Enterobacter agglomerans was weakly positive and the others were negative. Antibodies from hybridoma 9B12 reacted with all six E. coli tested and with Enterobacter cloacae; however, 9B12 stopped producing E. coli-specific antibody. The antibodies from five hybridomas produced antibodies which reacted with a majority of the bacteria tested. Additional monoclonal antibodies that can supplement antibodies from 6H2 are needed.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Charles William Kaspar
Kaspar, Charles William, "The use of monoclonal and polyclonal antibodies to identify Escherichia coli " (1986). Retrospective Theses and Dissertations. 9752.