US 2022/0018833 A1Published application

Method for re-using hapten-coated probe in an immunoassay

AssigneeAccess Medical Systems, Ltd.

InventorsRobert F. Zuk, Qing Xia, Jenchieh Wu

Life of this patent

Estimating the term…

Loading patent…

Abstract

The present invention is directed immunoassay methods, which re-use a hapten-immobilized test probe and reagents for quantitating an analyte in different samples, anywhere from about 3 to 20 times, while maintaining acceptable clinical assay performance. The methods use a dual antibody conjugate solution comprising an anti-hapten antibody and a capture antibody against the analyte in each cycle. After the completion of each cycle of reaction, the test probe is dipped in an acidic solution having pH about 1-4, to elute the immunocomplex formed on the probe and to regenerate the hapten-immobilized probe. The robustness of the hapten-coated solid phase allows utilization of multiple denaturation reagents for efficient elution of the immune complexes after each cycle without compromising the binding activity of the hapten on the solid phase.

Claims18

Dependent claims

A method of detecting an analyte in multiple liquid samples, comprising the steps of:

(a) dipping a probe in an aqueous solution having pH of 6.0-8.5 to pre-read the fluorescent signal of the probe tip, wherein the probe comprises a first hapten immobilized on the tip of the probe, and the diameter of the tip surface is ≤5 mm;
(b) forming a first immunocomplex comprising the analyte from a sample, a capture antibody, and a signal antibody on the probe tip, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-first hapten antibody against the first hapten and the signal antibody is conjugated with a second hapten, the first hapten and the second hapten are different;
(c) dipping the probe tip in a wash solution;
(d) dipping the probe tip in an amplification solution comprising an anti-second hapten antibody or streptavidin conjugated to fluorescent labels to form a second immunocomplex comprising the analyte, the capture antibody, the signal antibody, the second hapten, and the anti-second hapten antibody or streptavidin on the probe tip;
(e) determining the analyte concentration in the sample by measuring the fluorescent signal of the second immunocomplex at the probe tip, subtracting the pre-read fluorescent signal of (a), and quantitating the subtracted signal against a calibration curve;
(f) dipping the probe tip in an acidic solution having pH about 1.0-4.0 to elute the immunocomplexes from the probe tip;
and
(g) repeating the same steps (a)-(f) except in step (b) with the analyte from a new sample, whereby the analyte in each of the multiple liquid samples is detected.

5 more elements

A method of detecting an analyte in multiple liquid samples, comprising the steps of:

(a) obtaining a probe having a non-biotin hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm;
(b) forming a first immunocomplex comprising the analyte from a sample, a capture antibody, and a signal antibody on the probe tip, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-hapten antibody and the signal antibody is conjugated with biotin;
(c) dipping the probe tip in a wash solution;
(d) dipping the probe tip in an amplification solution comprising streptavidin conjugated to fluorescent labels to form a second immunocomplex comprising the analyte, the capture antibody, the signal antibody, the biotin, and streptavidin on the probe tip;
(e) reading a first fluorescent signal of the second immunocomplex at the probe tip;
(f) dipping the probe tip in an acidic solution having pH about 1.0-4.0;
(g) reading a second fluorescent signal of the acid-treated probe tip,
(h) subtracting the second fluorescent signal from the first fluorescent signal, and quantitating the subtracted signal against a calibration curve;
(i) dipping the probe tip in dimethyl sulfoxide;
and
(j) repeating the same steps (b)-(i) except in step (b) with a new sample solution comprising a new sample, whereby the analyte in each of the multiple liquid samples is detected.

8 more elements

A method of detecting an analyte in multiple liquid samples, comprising the steps of:

(a) dipping a probe in an aqueous solution having pH of 6.0-8.5 to pre-read the fluorescent signal of the probe tip, wherein the probe comprises a hapten immobilized on the tip of the probe, and the diameter of the tip surface is ≤5 mm;
(b) forming an immunocomplex comprising the analyte from a sample, a capture antibody, and a signal antibody on the probe tip, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-hapten antibody against the hapten and the signal antibody is conjugated with fluorescent labels;
(c) dipping the probe tip into a washing vessel containing a wash solution;
(d) reading a first fluorescent signal of the immunocomplex at the probe tip;
(e) dipping the probe tip in an acidic solution having pH about 1.0-4.0 and containing guanidium chloride;
(f) reading a second fluorescent signal of the acid-treated probe tip,
(g) subtracting the second fluorescent signal from the first fluorescent signal, and quantitating the subtracted signal against a calibration curve;
(h) dipping the probe tip in a chaotropic agent different from guanidium chloride;
and
(i) repeating the same steps (b)-(h) except in step (b) with a new sample solution comprising a new sample, whereby the analyte in each of the multiple liquid samples is detected.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application: 17/488,095Filed 28 Sep 2021
Priority: 3 Apr 2019
Published: 20 Jan 2022
Term ends: Not estimatedThe source records don’t support an estimate.

Classification7 codes

G01N 33/54313
With an insoluble carrier for immobilising immunochemicals: the carrier being characterised by its particulate form
G01N 21/6428
Fluorescence; Phosphorescence: measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g.…
G01N 21/645
Fluorescence; Phosphorescence: specially adapted constructive features of fluorimeters
G01N 21/8483
Systems specially adapted for particular applications: investigating reagent band