# US 10,119,967 B2 · Multiplex immuno screening assay

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- Number: US 10,119,967 B2
- Document ID: US10119967B2
- Status: Granted
- Kind: B2
- Application number: 14/396,841
- Filed: 2013-05-03
- Published: 2015-04-09
- Granted: 2018-11-06
- Earliest claimed: 2012-05-04
- Assignees: Institut Pasteur
- Applicants: Institut Pasteur
- Inventors: Jean-Claude Manuguerra; Jessica Vanhomwegen; Philippe Despres; Sylvie Paulous
- Web page: https://patentgrep.com/patent/US10119967B2

## CPC

- G01N 33/54306: {Solid-phase reaction mechanisms}
- G01N 33/543: with an insoluble carrier for immobilising immunochemicals
- G01N 33/54313: {the carrier being characterised by its particulate form}
- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- G01N 33/564: for pre-existing immune complex or autoimmune disease {, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9}
- G01N 33/56983: {Viruses}
- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- G02B 6/0016: {Grooves, prisms, gratings, scattering particles or rough surfaces}
- G02B 6/0038: {Linear indentations or grooves, e.g. arc-shaped grooves or meandering grooves, extending over the full length or width of the light guide}
- G02B 27/0081: {with means for altering, e.g. enlarging, the entrance or exit pupil}
- G02B 27/0172: {characterised by optical features}
- G02B 27/4272: {having plural diffractive elements positioned sequentially along the optical path}
- G01N 2333/18: Togaviridae; Flaviviridae
- G01N 2333/185: Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
- G01N 2469/20: Detection of antibodies in sample from host which are directed against antigens from microorganisms
- G02B 2027/0125: {Field-of-view increase by wavefront division}
- G02B 2027/0174: {holographic}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention provides kits and assay methods for the early detection of pathogens, precise identification of the etiologic agent, and improved disease surveillance. More specifically, the present invention discloses an immunoassay leading to the rapid and simultaneous detection of antibodies to a wide range of infectious pathogens in biological fluids of infected patients. This immunoassay involves the covalent and oriented coupling of fusion proteins comprising an AGT enzyme and a viral antigen on an identifiable solid support (e.g. fluorescent microspheres), said support being previously coated with an AGT substrate. This coupling is mediated by the irreversible reaction of the AGT enzyme on its substrate. The thus obtained antigen-coupled microspheres show enhanced capture of specific antibodies as compared to antigen-coupled microspheres produced by standard amine coupling procedures. The methods of the invention possess the ability to multiplex, minimize the amount of biological sample, and have enhanced sensitivity and specificity toward target antibodies as compared with classical ELISA or Radio-Immunoprecipitation assays.

## Claims

### Claim 1

An in vitro assay method for detecting at least two different target antibodies present in a biological sample from a subject, said method comprising the steps of:
(a) contacting a mixture of at least 2 solid supports with a biological sample from a subject comprising a mixture of human antibodies;
wherein one of the solid supports comprises a first 6-alkylguanine-DNA-alkyltransferase-Antigen fusion protein covalently coupled to a 6-alkylguanine-DNA-alkyltransferase-Antigen substrate, and
wherein another of the solid supports comprises a second 6-alkylguanine-DNA-alkyltransferase-Antigen fusion protein covalently coupled to a 6-alkylguanine-DNA-alkyltransferase-Antigen substrate;
(b) detecting the presence or absence of binding of antibodies to the first 6-alkylguanine-DNA-alkyltransferase-Antigen fusion protein by detecting the binding or lack of binding of one of the two target antibodies to the first fusion protein; and
(c) detecting the presence or absence of binding of antibodies to the second 6-alkylguanine-DNA-alkyltransferase-Antigen fusion protein by detecting the binding or lack of binding of the second of the two target antibodies to the second fusion protein.

### Claim 2 (depends on 1)

The assay method of claim 1, wherein a mixture of at least 15 solid supports is used in step (a).

### Claim 3 (depends on 1)

The assay method of claim 1, wherein both 6-alkylguanine-DNA-alkyltransferase-Antigen fusion proteins comprise the amino acid sequence of SEQ ID NO:2.

### Claim 4 (depends on 1)

The assay method of claim 1, wherein said solid supports are microparticles.

### Claim 5 (depends on 1)

The assay method of claim 1, wherein said solid supports are magnetic.

### Claim 6 (depends on 1)

The assay method of claim 1, wherein said solid supports are microparticles internally labeled with fluorescent dyes.

### Claim 7 (depends on 1)

The assay method of claim 1, wherein each of the solid supports emits a different and distinguishable wave length after excitation.

### Claim 8 (depends on 1)

The assay method of claim 1, wherein said first or second AGT-Antigen fusion protein comprises at least 20 consecutive amino acids of the viral EDIII protein of West-Nile virus.

### Claim 9 (depends on 1)

The assay method of claim 1, wherein said biological sample is serum or plasma.