A multiplexed, indirect enzyme-linked immunoassay for the detection and differentiation of E. coli from other Enterobacteriaceae and P. aeruginosa from other glucose non-fermenters

• Published in: Journal of microbiological methods 2019-03, Vol.158, p.52-58
• Main Authors: Jackson, N., Wu, T.Z., Adams-Sapper, S., Satoorian, T., Geisberg, M., Murthy, N., Lee, L., Riley, L.W.
• Format: Article
• Language: English
• Subjects: Antibodies, Bacterial - immunology; Bacterial detection; ELISA; Enterobacteriaceae - classification; Enterobacteriaceae - isolation & purification; Enzyme-Linked Immunosorbent Assay; Escherichia coli - immunology; Escherichia coli - isolation & purification; Fermentation; Glucose - metabolism; Gram-negative bacteria; Gram-Negative Bacterial Infections - microbiology; Humans; Latex agglutination test; Latex Fixation Tests; Microbial Sensitivity Tests; Pseudomonas aeruginosa - immunology; Pseudomonas aeruginosa - isolation & purification; Sensitivity and Specificity
• ISSN: 0167-7012; 1872-8359; 1872-8359
• DOI: 10.1016/j.mimet.2019.01.014

Gram-negative bacteria (GNB) are important causes of community (CA) and hospital (HA)- associated infections. Here we describe the development of an indirect ELISA (I-ELISA), which can be used to detect and differentiate the Enterobacteriaceae Escherichia coli, and glucose non-fermenter Pseudomonas aeruginosa from other GNB species. The I-ELISA utilizes six antibodies for bacterial speciation, which were grouped according to their bacterial targets; Enterobacteriaceae (SL-EntA and CH1810 mAb), Escherichia coli (SL-EcA and 6103–46 mAb), Pseudomonas aeruginosa (SL-PaA and SL-PaB). The six, anti-GNB antibodies were first screened against a panel of well-characterized clinical GNB isolates to optimize assay conditions and to determine individual antibody sensitivity and specificity. When tested against a diverse, blinded panel of 94 GNB clinical isolates, the I-ELISA exhibited the following sensitivity/specificity for each target: Enterobacteriaceae (94.4%/95%), E. coli (82.6%/88.7%), P. aeruginosa (83.3%/96%). An I–ELISA to detect and differentiate the most common GNB pathogens offers advantage in terms of simplicity over diagnostic tests currently used in most clinical settings. •Developed an indirect-ELISA against Enterobacteriaceae, E. coli and P. aeruginosa•Two antibodies were validated against each target organism.•The indirect-ELISA was both highly sensitive (≥ 82.6%) and specific (≥ 88.7%).•The validated antibodies could also be applied in a latex bead agglutination assay.