Rapid and high-sensitivity quantitative fluorescent immunoassay for detection of Klebsiella pneumoniae carbapenemase

IntroductionEarly and precise identification of Klebsiella pneumoniae carbapenemase (KPC) is critical for guiding effective treatment and curbing the spread of carbapenem-resistant pathogens. The mainstream detection methods targeting KPC are exclusively qualitative, lacking reliable quantitative approaches. To address the need for a rapid and quantitative method, this study developed a sensitive fluorescence immunoassay.MethodsThe assay employs a sandwich immunoassay format, which utilizes carboxylated magnetic agarose microspheres for capture and time-resolved fluorescent microspheres for detection. It enables quantification of the KPC protein within approximately 1 h. The analytical performance of the assay was systematically evaluated, including its sensitivity, linear range, specificity and accuracy.ResultsUnder optimal experimental conditions, it exhibits a wide linear range (0.25–1000 ng/mL) and a low detection limit of 0.194 ng/mL, and shows no cross-reactivity with other common carbapenemases. Recovery rates in bacterial lysates ranged from 96.7 to 108.4%. Compared to a commercial immunochromatographic test, this method demonstrated superior sensitivity.DiscussionThe primary advantage of this fluorescent immunoassay is its ability to achieve rapid, sensitive and quantitative detection of KPC, which addresses a key limitation of current qualitative methods. Its wide linear range and low detection limit enable precise monitoring of KPC expression level variations, offering a novel tool for the clinical assessment of bacterial resistance burden.