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Journal of Clinical Microbiology, June 2002, p. 1947-1957, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.1947-1957.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay for Detecting and Quantifying CMY-2 and SHV ß-Lactamases

Andrea M. Hujer,1 Malcolm G. P. Page,2 Marion S. Helfand,3 Bethany Yeiser,1 and Robert A. Bonomo1*

Research Service, Louis Stokes Veterans Affairs Medical Center, Cleveland, Ohio 44106,1 Basilea Pharmaceutica Ltd., Basel, Switzerland,2 Infectious Diseases Division, University Hospitals, Cleveland, Ohio 441063

Received 6 September 2001/ Returned for modification 31 October 2001/ Accepted 1 March 2002

Polyclonal rabbit antibodies against SHV-1 and CMY-2 ß-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 ß-lactamase tested. The anti-CMY-2 antibody detected Escherichia coli CMY-2, Enterobacter cloacae P99, Klebsiella pneumoniae ACT-1, and the AmpC ß-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 ß-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 ß-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC ß-lactamases of M. morganii, C. freundii, E. coli, and E. cloacae). As the homology to CMY-2 ß-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 ß-lactamases, and the results were confirmed with PCR amplification of blaAmpC and blaSHV genes. Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC ß-lactamases. The ELISA format can facilitate the identification of AmpC and SHV ß-lactamases and can be used to quantify relative amounts of ß-lactamase enzymes in clinical and laboratory isolates.

* Corresponding author. Mailing address: Infectious Disease, Louis Stokes Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4788. Fax: (216) 231-3482. E-mail: robert.bonomo{at}med.va.gov.

Journal of Clinical Microbiology, June 2002, p. 1947-1957, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.1947-1957.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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