Compositions and methods for determining successful immunization by one or more vaccines

• US 9,739,773 B1
• Filed: 02/17/2015
• Issued: 08/22/2017
• Est. Priority Date: 08/13/2010
• Status: Active Grant

• Current Assignee: David Gordon Bermudes
• Original Assignee: David Gordon Bermudes
• Inventors: Bermudes, David Gordon
• Primary Examiner(s): Andres, Janet L
• Assistant Examiner(s): Salvoza, M. Franco
• Application Number:
  US14/623,956
• Time in Patent Office: 917 Days
• Field of Search: None
• CPC Class Codes: G01N 2469/20 Detection of antibodies in sample from host which are directed against antigens from microorganisms
• CPC Class Codes: G01N 33/54388 based on lateral flow
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

A host antigen-specific antibody testing system and method. The a ternary complex of the antigen, a ligand-bound anti-host IgM, and a non-host anti-antigen IgG detector conjugate selectively form a quaternary complex with host antibodies, wherein the host antibodies and IgG compete for the antigen, and the anti-host IgM binds the host antibodies. The quaternary complex is retained by an immobilized IgM ligand binding agent, and any residual ternary complex is retained by a later encountered immobilized anti-non-host IgG. If sufficient host antibodies have a high affinity for the antigen, the complex is detected at the quaternary complex detection region based on the presence of the detector, and if there are insufficient high affinity host antibodies, the ternary complex migrates past the quaternary complex detection region and is retained and detected at a control region.

Claims

1. A testing method for presence of antibodies to an antigen from a host, comprising:

(a) providing;

(1) a reactant layer, into which are absorbed a ternary complex comprising;

the antigen,

a host antibody binding agent comprising at least one of an anti-host immunoglobulin IgM antibody and an anti-host immunoglobulin IgG antibody, bound to a ligand, and

a detector comprising a non-host anti-antigen IgG antibody conjugated to colloidal particles;

(2) a test strip, in fluid communication with the reactant layer at a portion thereof, to which is immobilized;

a ligand binding agent at a test area; and

an anti-non-host IgG antibody immobilized at a control area,

the test area being spaced more proximate to the reactant layer than the control area; and

(3) a sample well,

(b) placing a serum specimen from a host in the sample well;

(c) selectively forming a quaternary complex of;

host antigen-specific antibody in the serum specimen;

the detector;

the antigen; and

the host antibody binding agent;

(d) capturing the quaternary complex which migrates through the test strip using the immobilized ligand binding agent at the test area; and

(e) capturing the complex comprising the antigen and the detector, unbound to host antigen-specific antibodies, which migrates through the test strip past the test area, by using the immobilized anti-non-host IgG antibody,

to thereby indicate a presence of the host antigen-specific antibodies by an indication at the test area and validity of the test by an indication at the control area.

2. The method according to claim 1, wherein:

the ligand is biotin and the ligand binding agent is streptavidin;

the colloidal particles are colloidal gold particles;

the host antibody binding agent bound to a ligand comprises biotinylated goat-antihuman IgM antibody;

the non-host anti-antigen IgG antibody comprises mouse monoclonal anti-antigen IgG antibody;

the anti-non-host IgG antibody immobilized at the control area comprises rabbit anti-mouse IgG-Fc;

the reactant layer comprises a polyester pad;

the test strip comprises a nitrocellulose membrane;

the test strip comprises an elongated strip, on which the reactant layer is situated on one side;

the sample well is on top of the reactant layer;

a flow control layer is provided between the reactant layer and the test strip;

the test area and control area are sequentially disposed distant from the test strip; and

the antigen, the host antibody binding agent bound to a ligand, and the detector are provided in at least one solution,

further comprising;

providing an absorbent pad in fluid communication with the test strip disposed on an opposite side of the control area from the reactant layer, wherein the absorbent pad induces a bulk fluid flow from the reactant layer to the absorbent pad; and

dispensing and subsequently lyophilizing the at least one solution on the reactant layer.

3. A testing method for presence of antibodies to a particular antigen from a host, comprising:

providing a reactant layer, having the particular antigen, at least one of an anti-host immunoglobulin IgM antibody and an anti-host immunoglobulin IgG antibody bound to a ligand, and a non-host anti-antigen IgG antibody conjugated to a detector;

forming a complex by adding serum containing host antibodies to the particular antigen, wherein the host antibodies and the non-host anti-antigen IgG antibody bind the particular antigen, and the at least one of an anti-host immunoglobulin IgM antibody and an anti-host immunoglobulin IgG antibody bound to a ligand binds the host antibodies;

migrating the complex on a strip toward a first region having an immobilized ligand binding agent which binds and ceases migration of the at least one of an anti-host immunoglobulin IgM antibody and an anti-host immunoglobulin IgG antibody bound to a ligand and any complexes including the at least one of an anti-host immunoglobulin IgM antibody and an anti-host immunoglobulin IgG antibody bound to a ligand;

migrating an unbound residual portion of the complex past the first region to a second region having immobilized anti-non-host IgG antibody immobilized at a control area, which binds the non-host anti-antigen IgG antibody and any complexes including the non-host anti-antigen IgG antibody,

wherein,

if the host antibodies have a sufficient affinity for the antigen and are present in sufficient quantity, a complex comprising the antigen, the host antibodies, and the at least one of an anti-host immunoglobulin IgM antibody and an anti-host immunoglobulin IgG antibody bound to a ligand and the non-host anti-antigen IgG antibody conjugated to the and detector are retained in the first region by the immobilized ligand binding agent for detection based on the presence of the detector, and

if the host antibodies have insufficient affinity for the antigen or are not present in sufficient quantity, a complex comprising the antigen, and the non-host anti-antigen IgG antibody conjugated to the detector, will migrate past the first region and be retained at the second region by the immobilized anti-non-host IgG antibody for detection based on the presence of the detector.

20. The method according to claim 3, wherein:

the ligand is biotin and the ligand binding agent is streptavidin;

the non-host anti-antigen IgG antibody comprises a monoclonal antibody;

the anti-non-host IgG immobilized at the control area comprises an IgG-Fc;

the reactant layer is situated over a flow control layer on one side of a strip, with a sample well on top of the reactant layer;

the first region and the second region are sequentially disposed distant from the reactant layer; and

an absorbent pad is disposed on an opposite side of the second region from the reactant layer, which induces a bulk fluid flow from the reactant layer to the absorbent pad; and

the antigen, the at least one of the anti-host immunoglobulin IgM antibody and the anti-host immunoglobulin IgG antibody bound to the ligand, and the detector are lyophilized in the reactant layer.

4. A testing method for presence of host antigen-specific antibodies to an antigen from a host, comprising:

(a) providing;

(1) a reactant layer, into which are absorbed the antigen, an anti-host immunoglobulin antibody bound to a ligand, and a detector comprising a non-host anti-antigen antibody conjugated to colloidal particles;

(2) a test strip configured to permit migration of a fluid and to provide fluid communication with the reactant layer at a portion thereof, to which is immobilized a ligand binding agent at a test area and anti-non-host antibody immobilized at a control area, the test area being spaced more proximate to the reactant layer than the control area; and

(3) a sample well in fluid communication with the test strip at a portion thereof,

(b) placing a fluid serum specimen from a host in the sample well, and permitting the fluid serum specimen to migrate into and through the test strip;

(c) permitting host antigen-specific antibodies in the fluid serum specimen to selectively form a quaternary host antigen-specific antibody/detector/antigen/anti-host immunoglobulin antibody complex with the detector, antigen, and anti-host immunoglobulin antibody;

(d) capturing quaternary host antigen-specific antibody/detector/antigen/anti-host immunoglobulin antibody complex which migrates through the test strip using the immobilized ligand binding agent at the test area; and

(e) capturing the detector and antigen, unbound to antigen-specific host antibodies, which migrates through the test strip past the test area using the immobilized anti-non-host antibody,

to thereby indicate a presence of the host antigen-specific antibodies by an indication at the test area and validity of the test by an indication at the control area.

5. The method according to claim 4, wherein the ligand is biotin and the ligand binding agent is streptavidin.

6. The method according to claim 4, wherein the colloidal particles are gold particles.

7. The method according to claim 4, wherein anti-host immunoglobulin antibody bound to the ligand is an anti-host immunoglobulin IgM-antibody bound to the ligand, and the concentrations of antigen, anti-host immunoglobulin IgM-antibody bound to the ligand, the detector, immobilized ligand binding agent and immobilized anti-non-host antibody are provided, based on calibration with pooled sera from other hosts which have responded to an immunization with the antigen by production of the host antigen-specific antibodies, to produce a positive indication at an IgM Index value of at least 1.1 of an IgM Capture ELISA device and a negative indication results below an IgM Index value of 1.1 of the IgM Capture ELISA device.

8. The method according to claim 4, wherein the antigen is an influenza hemagglutinin.

9. The method according to claim 4, wherein the antigen is an influenza hemagglutinin produced by the genetically engineered bacterium.

10. The method according to claim 4, wherein the antigen is a glycosylated antigen produced in an insect cell/baculovirus system.

11. The method according to claim 4, wherein the anti-host immunoglobulin antibody bound to a ligand comprises biotinylated goat-antihuman IgM antibody.

12. The method according to claim 4, wherein the non-host anti-antigen antibody comprises a mouse monoclonal IgG anti-antigen antibody.

13. The method according to claim 12, wherein the anti-non-host antibody immobilized at the control area comprises a rabbit anti-mouse IgG-Fc.

14. The method according to claim 12, wherein the mouse monoclonal IgG anti-antigen antibody is conjugated onto colloidal gold particles.

15. The method according to claim 4, wherein the antigen, the anti-host immunoglobulin antibody bound to the ligand, and the detector are dispensed as at least one solution on the reactant layer and subsequently lyophilized.

16. The method according to claim 4, wherein the reactant layer comprises a polyester pad.

17. The method according to claim 4, wherein the test strip comprises a nitrocellulose membrane.

18. The method according to claim 4,

wherein the test strip comprises an elongated strip, on which the reactant layer is situated on one-side of the test strip, and the test area and control area are sequentially disposed distant from the test strip along a migration path of the fluid through the test strip, the sample well is on top of the reactant layer, and between the reactant layer and the test strip is disposed at least one flow control layer,

further comprising:

(f) providing an absorbent pad in fluid communication with the test strip disposed on an opposite side of the control area from the reactant layer with respect to the migration path of the fluid through the test strip; and

(g) inducing a bulk migration of fluid from the reactant layer to the absorbent pad.

19. The method according to claim 4, wherein:

the ligand is biotin and the ligand binding agent is streptavidin;

the colloidal particles are colloidal gold particles;

the anti-host immunoglobulin antibody bound to a ligand comprises biotinylated goat-antihuman IgM antibody;

the non-host anti-antigen antibody comprises mouse monoclonal anti-antigen IgG antibody; and

the anti-non-host antibody immobilized at the control area comprises rabbit anti-mouse IgG-Fc,

further comprising;

(f) providing an absorbent pad in fluid communication with the test strip disposed on an opposite side of the control area from the reactant layer with respect to a path of fluid migration through the test strip, wherein the absorbent pad induces a bulk fluid flow from the reactant layer to the absorbent pad; and

(g) dispensing as at least one solution and subsequently lyophilizing the antigen, the anti-host immunoglobulin antibody bound to the ligand, and the detector on the reactant layer,

wherein the test strip comprises an elongated strip, on which the reactant layer is situated on one side, the test area and control area are sequentially disposed along a fluid migration path from the sample well in the test strip, and the sample well is on top of the reactant layer, and between the reactant layer and the test strip is disposed at least one flow control layer.