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J Microbiol Methods. 2018 Jan;144:37-43. doi: 10.1016/j.mimet.2017.09.017. Epub 2017 Sep 29.

Monoclonal antibody-mediated detection of CTX-M β-lactamases in Gram-negative bacteria.

Author information

1
School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, CA 94720, USA.
2
Silver Lake Research Corporation, Azusa, CA 91702, USA.
3
School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, CA 94720, USA. Electronic address: lwriley@berkeley.edu.

Abstract

Gram-negative bacteria (GNB) that express CTX-M β-lactamases have become a serious threat to the clinical management of GNB infections. While antibody-based platforms have been successfully used in research settings to study and detect other β-lactamases-including SHV, CMY, and TEM enzymes-there is currently a lack of antibody-based tools to detect the CTX-M enzymes. Here we describe the development of an anti-CTX-M sandwich ELISA based on a pair of monoclonal antibodies (mAbs)-mAb 6101-33 and mAb 6101-19-used as the capture and detection antibody, respectively. This antibody pair detected CTX-M variants from group 1 (CTX-M-15), group 2 (CTX-M-2), group 8 (CTX-M-8), and group 9 (CTX-M-14) that were expressed by a training set of clinical GNB isolates. The limit of detection for this sandwich ELISA was 30ng of recombinant CTX-M-15, and CTX-Ms expressed by 106 lysed CFU of GNB. When tested against a blinded panel of 78 clinical isolates, the sandwich ELISA demonstrated a sensitivity of 96% and a specificity of 100%. The mAb pair did not cross-react with bacteria that contained other β-lactamases, including TEM, SHV, OXA, KPC, NDM, CMY, and DHA. In conclusion, we developed a highly sensitive and specific sandwich ELISA, capable of detecting CTX-M enzyme production in GNB pathogens.

KEYWORDS:

CTX-M; ELISA; ESBL; Gram-negative bacteria; β-lactamase detection

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