Haptens and conjugates derived from pyocyanin, antibodies thereof, and immunochemical method for detecting infections caused by Pseudomonas aeruginosa
Inventors
MARCO COLAS, MARIA PILAR • Pascual Duran, Nuria • Pastells Diez, Carme • Sanchez Baeza, Francisco • Villaverde Corrales, Antonio Pedro • Rodriguez Carmona, Escarlata
Assignees
Consejo Superior de Investigaciones Cientificas CSIC • Universitat Autonoma de Barcelona UAB • Centro de Investigacion Biomedica en Red CIBER
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10422791-B2
Publication Date
2019-09-24
Expiration Date
Abstract
The present invention relates to a compound of general formula I and to the use thereof as a hapten.An object of the present invention is also a conjugate of said compound I with a carrier protein or fragment thereof, with a detectable labelling agent, or with a polymer or support, and to the use thereof for producing antibodies. Furthermore, the present invention also relates to a method for the detection and/or quantification of 1-hydroxyphenazine and/or pyocyanin using said antibodies and conjugates for the detection of infections caused by Pseudomonas aeruginosa.
Core Innovation
The invention relates to haptens related to pyocyanin/phenazine for immunochemical detection of Pseudomonas aeruginosa and to ELISA formats for detecting phenazine analytes, including 1-hydroxyphenazine and pyocyanin, using immobilized phenazine hapten conjugates. It provides hapten compounds according to a general formula I for use in immunochemistry, and a conjugate in which a hapten comprising a mixture of 9-hydroxyphenazine-2-carboxylic acid and 6-hydroxyphenazine-2-carboxylic acid is attached to a carrier protein.
The document describes an indirect competitive ELISA approach using a phenazine-specific primary antibody and an enzyme-labeled secondary antibody, preferably an anti-IgG-HRP, to generate a detectable signal. The assay is based on competitive binding in which the hapten identity with the antibodies is used as the competitive criterion for determining phenazine analyte levels, with signal generation described using chromogenic, fluorogenic, or chemiluminescent substrates.
The document additionally describes immunochemical components and workflows including antibodies generated using the conjugates, together with detectable labels and immunoassay formats such as ELISA. It also addresses detection and quantification in vitro, including converting pyocyanin to 1-hydroxyphenazine prior to the competitive assay, and discusses analytical performance, specificity/cross-reactivity, matrix effects, and recovery in sputum and plasma.
A kit/device concept is also described for field or minimally automated use of the ELISA workflow for phenazine detection and quantification. The partial content further indicates extensive experimental support focused on conjugate/antibody preparation overview, selection of an optimal antiserum/bioconjugate pair, and validation of conversion and analytical behavior in biological matrices.
Claims Coverage
The provided independent claims cover a conjugate defined by a specific hapten mixture and attachment to a carrier protein, with dependent claims refining the carrier protein selection. The inventive features are centered on the hapten mixture composition and the carrier protein selection.
Hapten mixture of 9-hydroxyphenazine-2-carboxylic acid and 6-hydroxyphenazine-2-carboxylic acid
A hapten comprising a mixture of 9-hydroxyphenazine-2-carboxylic acid and 6-hydroxyphenazine-2-carboxylic acid.
Carrier protein attached to the hapten mixture
A conjugate comprising the hapten mixture and a carrier protein.
Carrier protein selection from named proteins
The carrier protein is selected from horseshoe crab hemocyanin (HCH), bovine serum albumin (BSA), horseradish peroxidase (HRP), ovalbumin (OVA), or conalbumin (CONA).
Overall, the claims cover a hapten-carrier conjugate in which the hapten is a mixture of 9-hydroxyphenazine-2-carboxylic acid and 6-hydroxyphenazine-2-carboxylic acid, with dependent coverage specifying a particular named set of carrier proteins.
Stated Advantages
Enables immunochemical detection of pyocyanin/phenazine related analytes associated with Pseudomonas aeruginosa.
Supports immunochemical measurement workflows by providing hapten-carrier conjugates used to generate antibodies and perform immunoassays such as ELISA.
Documented Applications
Immunochemical detection of Pseudomonas aeruginosa using haptens related to pyocyanin/phenazine.
In vitro detection/quantification workflows for 1-hydroxyphenazine and detection of pyocyanin via conversion to 1-hydroxyphenazine.
ELISA-based immunoassay using an example conjugate (e.g., PC1-BSA) and rabbit antiserum (e.g., As230), including characterization of assay performance (calibration curves, precision, LOD/IC50/IC90, cross-reactivity, matrix effects, and recovery in sputum/plasma).
Field or minimally automated use of the ELISA workflow for phenazine detection and quantification.
Interested in licensing this patent?