# US 8,148,057 B2 · Methods, immunoassays and devices for detection of anti-lipoidal antibodies

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- Number: US 8,148,057 B2
- Document ID: US8148057B2
- Status: Granted
- Kind: B2
- Application number: 12/433,626
- Filed: 2009-04-30
- Published: 2009-10-22
- Granted: 2012-04-03
- Earliest claimed: 2005-06-21
- Assignees: The United States of America as represented by the Secretary of the Department of Health and Human Services, Centers for Disease Control and Prevention
- Inventors: Arnold R. Castro
- Web page: https://patentgrep.com/patent/US8148057B2

## CPC

- G01N 33/571: for venereal disease, e.g. syphilis, gonorrhoea {(herpes G01N33/56994; chlamydia G01N33/56927)}
- G01N 33/5432: {Liposomes or microcapsules}
- G01N 33/54366: {Apparatus specially adapted for solid-phase testing}
- G01N 33/54388: {based on lateral flow}
- G01N 33/54391: {based on vertical flow}
- G01N 2333/20: from Spirochaetales (O), e.g. Treponema, Leptospira
- 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

Compositions, methods and devices for the detection of anti-lipoidal antibodies and the diagnosis of disease, for example, syphilis, are described. In particular, a method for immobilizing a lipoidal antigen, comprising cardiolipin, lecithin, and cholesterol, on a solid support (such as a nitrocellulose membrane) is described. The ability to immobilize a lipoidal antigen on a membrane satisfies a long-felt need for a membrane-based assay for the detection of anti-lipoidal antibodies. Also described are immunoassay devices for concurrently performing treponemal and non-treponemal tests for syphilis.

## Claims

### Claim 1

A method of making an immunoassay device for detection of anti-lipoidal antibodies comprising:
preparing a lipoidal antigen in a non-aqueous solution;
mixing the lipoidal antigen in a non-aqueous solution with an aqueous solution to generate lipoidal micelles;
centrifuging the lipoidal micelles to generate a supernatant comprising small lipoidal antigen micelles having an average diameter of less than 5 microns;
applying the small lipoidal antigen micelles to the microporous membrane having a proximal sample application area and a detection zone with a predetermined pore size, wherein the small antigen micelles enter and are retained in the pores of the detection zone, thereby forming an immunoassay device for detection of anti-lipoidal antibodies.

### Claim 2 (depends on 1)

The method of claim 1, wherein the immunoassay device is a flow-through or lateral flow device and the small lipoidal antigen micelles are applied in a linear distribution to form a test line.

### Claim 3 (depends on 1)

The method of claim 1, further comprising washing the microporous membrane to remove unbound small lipoidal antigen micelles from the microporous membrane.

### Claim 4 (depends on 1)

The method of claim 1, wherein the lipoidal antigen comprises a USR antigen, a VDRL antigen, a synthetic VDRL antigen or cardiolipin, lecithin and cholesterol.

### Claim 5 (depends on 1)

The method of claim 1, wherein the lipoidal antigen comprises from about 0.001% to about 0.1% cardiolipin; from about 0.05% to about 0.5% lecithin; and/or from about 0.2% to about 5% cholesterol.

### Claim 6 (depends on 1)

The method of claim 1, wherein the lipoidal antigen comprises cardiolipin, lecithin and cholesterol.

### Claim 7 (depends on 1)

The method of claim 1, wherein the non-aqueous solution to aqueous solution is present as a ratio of about 1:10, about 1:15, about 2:33, or about 1:20.

### Claim 8 (depends on 1)

The method of claim 1, wherein the small lipoidal antigen micelles comprise an average diameter of less than about 3 microns, less than about 1 micron, less than about 0.9 microns, less than about 0.8 microns, less than about 0.7 microns, less than about 0.6 microns, less than about 0.5 microns, less than about 0.4 microns, less than about 0.3 microns, or less than about 0.2 microns.

### Claim 9 (depends on 1)

The method of claim 1, wherein the microporous membrane comprises nitrocellulose, nylon, polyvinylidene fluoride (PVDF), polyethersulfone, polycarbonate, polyester, cellulose acetate, mixed cellulose esters, or combinations thereof; and, wherein the microporous membrane comprises a predetermined pore size with an average diameter from about 0.22 micron to about 20 microns.

### Claim 10 (depends on 1)

The method of claim 1, wherein the microporous membrane comprises a predetermined pore size with an average diameter from about 0.22 micron to about 0.45 micron, from about 0.22 micron to about 0.65 micron, from about 0.22 micron to about 0.8 micron, from about 0.22 micron to about 1.2 micron, from about 0.65 micron to about 3 microns, from about 0.65 micron to about 5 microns, from about 0.65 micron to about 8 microns, from about 5 microns to about 10 microns, or from about 5 microns to about 20 microns.

### Claim 11 (depends on 1)

An immunoassay device made by the method of claim 1.

### Claim 12 (depends on 11)

The immunoassay device of claim 11, for determining the presence or amount of an anti-lipoidal antibody in a liquid sample, further comprising a flow path from the sample application area to the detection zone; wherein the presence or amount of the anti-lipoidal antibody in the liquid sample is detected by formation of an immunocomplex in the detection zone between the anti-lipoidal antibody in the liquid sample and the retained small lipoidal antigen micelles.

### Claim 13 (depends on 12)

The immunoassay device of claim 12, wherein the device is a lateral flow device and wherein the lateral flow device further comprises a conjugate pad located in the flow path, wherein the conjugate pad comprises a mobile or mobilizable detector reagent specific for the anti-lipoidal antibody.

### Claim 14 (depends on 12)

The immunoassay device of claim 12, wherein the detector reagent comprises gold-conjugated Protein A, gold-conjugated Fc-specific Protein G, or gold-conjugated anti-human antibody (Fc portion).

### Claim 15 (depends on 13)

The immunoassay device of claim 13, wherein the conjugate pad further comprises a mobile or mobilizable detector reagent specific for an anti-T. pallidum antibody or a mobile treponemal antigen.

### Claim 16 (depends on 15)

The immunoassay device of claim 15, wherein the detector reagent specific for the anti-T. pallidum antibody comprises gold-conjugated Protein A, gold-conjugated Fc-specific Protein G, or gold-conjugated anti-human antibody(Fc portion), or the detector reagent for the mobile treponemal antigen comprises gold-labeled anti-treponemal antigen antibody.

### Claim 17 (depends on 11)

A method for detecting anti-lipoidal antibodies in a subject, comprising,
applying a biological sample from a subject to the immunoassay device of claim 11; and,
detecting formation of an immunocomplex between an anti-lipoidal antibody present in the biological sample and the immobilized small lipoidal antigen micelles in the detection zone of the immunoassay device, wherein detection of formation of the immunocomplex in the detection zone indicates the presence of the anti-lipoidal antibody in the subject.

### Claim 18 (depends on 17)

The method of claim 17, wherein the presence of the anti-lipoidal antibody in the subject is used to diagnose syphilis in the subject.

### Claim 19 (depends on 17)

The method of claim 17, wherein the biological sample is blood or serum.

### Claim 20 (depends on 1)

The method of claim 1, wherein the non-aqueous solution comprises ethanol.

### Claim 21 (depends on 1)

The method of claim 1, wherein the non-aqueous solution comprises a saline solution.

### Claim 22 (depends on 1)

The method of claim 1, wherein after the mixing step, the method further comprises:
centrifuging the lipoidal micelles to generate a pellet; and
resuspending the pellet in a buffer comprising 3.72% EDTA, 40% choline chloride (v/v), phosphate buffered saline pH 6.9 ±0.1, wherein centrifuging the lipoidal micelles comprises centrifuging the resuspended pellet.

### Claim 23 (depends on 1)

The method of claim 1, wherein centrifuging the lipoidal micelles generates a pellet comprising large lipoidal antigen micelles having an average diameter of greater than 5 microns, and the method further comprises reducing the large lipoidal antigen micelles to small lipoidal antigen micelles having an average diameter of less than 5 microns.

### Claim 24 (depends on 23)

The method of claim 23, wherein reducing the large lipoidal antigen micelles comprises filtration, sonication, differential centrifugation or a combination thereof, of the large lipoidal antigen micelles.

### Claim 25

A method of making an immunoassay device for detection of anti-lipoidal antibodies comprising:
preparing a synthetic VDRL antigen in a non-aqueous solution comprising ethanol;
mixing the synthetic VDRL antigen in a non-aqueous solution with an aqueous solution comprising saline solution to generate lipoidal micelles;
centrifuging the lipoidal micelles to generate a pellet;
resuspending the pellet in a buffer comprising 3.72% EDTA, 40% choline chloride (v/v), phosphate buffered saline pH 6.9 ±0.1, thereby generating a resuspended pellet;
centrifuging the resuspended pellet to generate a supernatant comprising small lipoidal antigen micelles having an average diameter of less than 5 microns;
applying the small lipoidal antigen micelles to the microporous membrane having a proximal sample application area and a detection zone with a pore size of 5 microns to about 10 microns, wherein the small lipoidal antigen micelles enter and are retained in the pores of the detection zone, thereby forming an immunoassay device for detection of anti-lipoidal antibodies.

### Claim 26 (depends on 25)

An immunoassay device made by the method of claim 25.