# US 12,111,312 B2 · Coronavirus IgG/IgM multiplexed dual path immunoassay device

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- Number: US 12,111,312 B2
- Document ID: US12111312B2
- Status: Granted
- Kind: B2
- Application number: 17/316,363
- Filed: 2021-05-10
- Published: 2021-11-11
- Granted: 2024-10-08
- Earliest claimed: 2020-05-11
- Assignees: Chembio Diagnostic Systems, Inc.
- Applicants: Chembio Diagnostic Systems, Inc.
- Inventors: Javanbakhsh Esfandiari
- Web page: https://patentgrep.com/patent/US12111312B2

## CPC

- G01N 33/54366: {Apparatus specially adapted for solid-phase testing}
- G01N 33/54389: {with bidirectional or multidirectional lateral flow, e.g. wherein the sample flows from a single, common sample application point into multiple strips, lanes or zones}
- G01N 33/56983: {Viruses}
- G01N 2333/165: Coronaviridae, e.g. avian infectious bronchitis virus
- G01N 2469/20: Detection of antibodies in sample from host which are directed against antigens from microorganisms
- 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)

## Abstract

Test cells with first and second sorbent materials defining a first flow path for a solution, a second flow path distinct from the first flow path for a sample, and a test site with immobilized antigens or antibodies or other ligand-binding molecules located at the junction of the sorbent materials for identifying one or more ligands. In one embodiment, a single highly sensitive immunoassay device is provided that detects the presence in a body fluid sample of two or more COVID-19 (Coronavirus disease 2019) antibodies including immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies to nucleocapsid protein (NP) and spike protein receptor binding domain (RBD), and optionally spike protein S1 subunit (S1) COVID-19 virus antigens. The immunoassay device is sensitive in detecting early infection using IgM antibody detection and continuing infection using IgG antibody detection. Additionally, successful inoculation is distinguished from infection after inoculation by comparing NP and RBD results.

## Claims

### Claim 1

A test device for determining presence of at least two different antibodies to a COVID-19 (Severe acute respiratory syndrome coronavirus-2) virus in a liquid sample, comprising:
a first test zone configured to detect presence of the at least two different antibodies to the COVID-19 virus;
a first sorbent strip having a first location for receiving a solution and defining a first migration path leading to the first test zone for the solution, wherein the first sorbent strip contains a first marker conjugate adapted to move with the solution along said first migration path and bind to said at least two different antibodies to the COVID-19 virus; and
a second sorbent strip distinct from said first sorbent strip and having a second location for receiving the liquid sample and defining a second migration path leading to the first test zone for the liquid sample, said first and second sorbent strips touching each other at said first test zone;
wherein the first test zone has first and second test sites located on or in at least one of said first sorbent strip and said second sorbent strip, said first test site having an immobilized first ligand binding mechanism that binds to antibodies to a first protein of the COVID-19 virus and said second test site having an immobilized second ligand binding mechanism that binds to antibodies to a second protein of the COVID-19 virus, wherein said second location is removed from said first and second test sites such that the liquid sample applied to said second location requires time to migrate to said first and second test sites and does not immediately wet said first and second test sites.

### Claim 2 (depends on 1)

The test device according to claim 1, wherein the test device determines the presence of at least three different antibodies to the COVID-19 virus and the first test zone includes a third test site located on or in one at least one of said first sorbent strip and said second sorbent strip adjacent the second test site, said third test site having an immobilized third ligand binding mechanism that binds to antibodies to a third protein of the COVID-19 virus.

### Claim 3 (depends on 1)

The test device according to claim 1, wherein the immobilized first and second ligand binding mechanisms of said first and second test sites include antigens that bind to IgG antibodies to a nucleocapsid protein (NP) and a spike protein receptor binding domain (RBD) of the COVID-19 virus.

### Claim 4 (depends on 1)

The test device according to claim 1, wherein the immobilized first and second ligand binding mechanisms of said first and second test sites include antigens that bind to IgM antibodies to a nucleocapsid protein (NP) and spike protein receptor binding domain (RBD) of the COVID-19 virus.

### Claim 5 (depends on 2)

The test device according to claim 2, wherein the immobilized first, second, and third ligand binding mechanisms of said first, second, and third test sites include antigens that bind to IgG antibodies to a nucleocapsid protein (NP) and a spike protein S1 subunit (S1) and a spike protein receptor binding domain (RBD) of the COVID-19 virus.

### Claim 6 (depends on 2)

The test device according to claim 2, wherein the immobilized first, second, and third ligand binding mechanisms of said first, second, and third test sites include antigens that bind to IgM antibodies to a nucleocapsid protein (NP) and a spike protein S1 subunit (S1) and a spike protein receptor binding domain (RBD) of the COVID-19 virus.

### Claim 7 (depends on 1)

The test device according to claim 1, further comprising a third sorbent strip which receives the solution and defines a third migration path leading to a second test zone for the solution, wherein the third sorbent strip contains a second marker conjugate different than said first marker conjugate, the second marker conjugate adapted to move with the solution along said third migration path and bind to antibodies to the COVID-19 virus, wherein the second sorbent strip touches the third sorbent strip at the second test zone, and wherein the second test zone has a plurality of additional test sites with different immobilized ligand binding mechanisms that bind to antibodies to different proteins of the COVID-19 virus.

### Claim 8 (depends on 7)

The test device according to claim 7, wherein the immobilized first and second ligand binding mechanisms of said first test zone includes antigens that bind to IgG antibodies to a nucleocapsid protein (NP) and a spike protein receptor binding domain (RBD) of the COVID-19 virus and the different immobilized ligand mechanisms of the additional test sites of the second test zone includes antigens that bind to IgM antibodies to a nucleocapsid protein (NP) and a spike protein receptor binding domain (RBD) of the COVID-19 virus.

### Claim 9 (depends on 7)

The test device according to claim 7, wherein the test device determines the presence of at least three different antibodies to the COVID-19 virus and the first test zone includes a third test site located on or in one at least one of said first sorbent strip and said second sorbent strip adjacent the second test site, said third test site having an immobilized third ligand binding mechanism that binds to antibodies to a third protein of the COVID-19 virus, and wherein the immobilized first, second, and third ligand binding mechanisms of the first, second and third test sites of said first test zone includes antigens that bind to IgG antibodies to a nucleocapsid protein (NP) and a spike protein S1 subunit (S1) and a spike protein receptor binding domain (RBD) of the COVID-19 virus, and the different immobilized ligand binding mechanisms of the additional test sites of the second test zone includes antigens that bind to IgM antibodies to a nucleocapsid protein (NP) and a spike protein S1 subunit (S1) and a spike protein receptor binding domain (RBD) of the COVID-19 virus.

### Claim 10 (depends on 1)

The test device according to claim 1, wherein the second sorbent strip includes a depletion zone located between said second location and said first test zone, wherein said depletion zone has depletion molecules or conjugates for broadly depleting one of COVID-19 virus IgG antibodies and COVID-19 virus IgM antibodies.

### Claim 11 (depends on 1)

The test device according to claim 1, wherein the second sorbent strip includes a depletion zone located between said second location and said first test zone, wherein said depletion zone has depletion molecules or conjugate comprising antigens or conjugates for depleting Human Coronavirus HKU1, 229E, NL63, and OC43 antibodies.

### Claim 12 (depends on 1)

The test device according to claim 1, wherein the first test zone includes a third test site located on or in one at least one of said first sorbent strip and said second sorbent strip adjacent the second test site and a fourth test site located on or in one at least one of said first sorbent strip and said second sorbent strip adjacent the third test site, said third test site having an immobilized third ligand binding mechanism that binds to antibodies to a third protein of the COVID-19 virus, and the fourth test site having an immobilized fourth ligand binding mechanism that binds to antibodies to a fourth protein of the COVID-19 virus, wherein said first, second, third and fourth ligand binding mechanisms include antigens that bind to IgG antibodies or IgM antibodies to a nucleocapsid protein (NP) and a spike protein S1 subunit (S1) and a spike protein S2 subunit (S2) and a spike protein receptor binding domain (RBD) of the COVID-19 virus.

### Claim 13 (depends on 1)

The test device according to claim 1, wherein the immobilized first and second ligand binding mechanisms of said first test zone include antigens that capture a plurality of COVID-19 IgG antibodies or COVID-19 IgM antibodies.

### Claim 14 (depends on 13)

The test device according to claim 13, wherein the antigens of said first test site include antigens selected from recombinant nucleocapsid proteins (NP), recombinant spike protein receptor binding domains (RBD), recombinant spike proteins (S1), and combinations thereof, and wherein the antigens of said second test site include antigens selected from recombinant nucleocapsid proteins (NP), recombinant spike protein receptor binding domains (RBD), and recombinant spike proteins (S1), and combinations thereof.

### Claim 15 (depends on 1)

The test device according to claim 1, wherein the first marker conjugate comprises a monoclonal anti-IgM gold conjugate or a Protein-A Gold conjugate.