Capture ELISA for KPC Detection in Gram-Negative Bacilli: Development and Standardisation

Abstract

The detection of KPC-type carbapenemases is necessary for guiding appropriate antibiotic therapy and the implementation of antimicrobial stewardship and infection control measures. Currently, few tests are capable of differentiating carbapenemase types, restricting the lab reports to their presence or not. The aim of this work was to raise antibodies and develop an ELISA test to detect KPC-2 and its D179 mutants. The ELISA-KPC test was designed using rabbit and mouse polyclonal antibodies. Four different protocols were tested to select the bacterial inoculum with the highest sensitivity and specificity rates. The standardisation procedure was performed using 109 previously characterised clinical isolates, showing 100% of sensitivity and 89% of specificity. The ELISA-KPC detected all isolates producing carbapenemases, including KPC variants displaying the ESBL phenotype such as KPC-33 and -66.

Detection of Microorganisms Using Artificial Siderophore–FeIII Complex-Modified Substrates

“…In addition, the process is complicated and requires expertise and skill, and viable and dead bacteria cannot be distinguished easily. Immunoassays − and enzyme-linked immunosorbent assays , utilize antibody–antigen reactions that are highly specific and sensitive, although the molecular elements required vary with the target microorganisms and cannot sufficiently respond to the structural changes caused by mutations. Other detection and identification methods for microorganisms, such as bacteria and fungi, utilize weak interactions with specific peptides, − sugar chains, proteins, − and biofilms on the cell surface; hence, the interacting molecules must be chosen according to the partner.…”

Urchin-Shaped Au–Ag@Pt Sensor Integrated Lateral Flow Immunoassay for Multimodal Detection and Specific Discrimination of Clinical Multiple Bacterial Infections

“…Bacterial infections pose a serious health threat to humans that urgently demands the development of fast and sensitive diagnostic methods . Traditional methods for detecting bacteria primarily rely on bacterial culturing, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), while they require complicated sample pretreatment and professional technical users, , which hinder their applications for point-of-care (POC) detection. Presently, numerous approaches have been established for the effective determination of bacteria such as lateral flow immunoassay (LFIA), electrochemical biosensor, and optical biosensor, − among which LFIA could satisfy all of the requirements for POC onsite detection.…”