title: Development of a sensitive and specific enzyme-linked immunosorbent assay for detecting and quantifying CMY-2 and SHV beta-lactamases.
abstract: Polyclonal rabbit antibodies against SHV-1 and CMY-2 beta-lactamases were produced and characterized, and enzyme-linked immunosorbent assays (ELISAs) were developed. Immunoblots revealed that the anti-SHV-1 antibody recognized SHV-1 but did not recognize TEM-1, K-1, OXA-1, or any AmpC beta-lactamase tested. The anti-CMY-2 antibody detected Escherichia coli CMY-2, Enterobacter cloacae P99, Klebsiella pneumoniae ACT-1, and the AmpC beta-lactamases of Enterobacter aerogenes, Morganella morganii, and Citrobacter freundii. No cross-reactivity of the anti-CMY-2 antibody was seen against laboratory strains of E. coli possessing TEM-1, SHV-1, K-1, or OXA-1 beta-lactamases. Operating conditions for performing ELISAs were optimized. Both anti-CMY-2 and anti-SHV-1 antibodies detected picogram quantities of purified protein in ELISAs. The reactivity of the anti-CMY-2 antibody was tested against a number of AmpC beta-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC beta-lactamases of M. morganii, C. freundii, E. coli, and E. cloacae). As the homology to CMY-2 beta-lactamase decreased, the minimum level needed for detection increased (e.g., 94% homology recognized at 1 ng/ml and 71% homology recognized at 10 ng/ml). The ELISAs were used to assay unknown clinical isolates for AmpC and SHV beta-lactamases, and the results were confirmed with PCR amplification of bla(AmpC) and bla(SHV) genes. Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC beta-lactamases. The ELISA format can facilitate the identification of AmpC and SHV beta-lactamases and can be used to quantify relative amounts of beta-lactamase enzymes in clinical and laboratory isolates.
authors.affiliation: Research Service, Louis Stokes Veterans Affairs Medical Center, Cleveland, Ohio 44106, USA.
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hujer
authors.forename: Malcolm G P
authors.initials: MG
authors.lastname: Page
authors.forename: Marion S
authors.initials: MS
authors.lastname: Helfand
authors.forename: Bethany
authors.initials: B
authors.lastname: Yeiser
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000276:immunology*; D001426:Bacterial Proteins; D004755:Enterobacteriaceae / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; C103022:beta-lactamase CMY-2; C048795:AmpC beta-lactamases; D001618:beta-Lactamases