BACKGROUND OF THE INVENTION

Immunoassays are frequently used to identify infectious agents, among other uses. Certain immunoassays rely on host immunological responses to a given infectious agent, for instance, by testing for the presence of host antibodies that specifically bind to one or more unique antigens of that infectious agent. Numerous types of immunoassay systems are available for diagnostic purposes, including large, automated central lab systems and relatively simple over-the-counter tests. These immunoassays utilize a broad range of test formats, such as agglutination assays, precipitin assays, enzyme-linked immunoassays, direct fluorescence assays, immuno-histological tests, complement-fixation assays, serological tests, immuno-electrophoretic assays, and lateral flow and flow through tests (i.e., rapid “strip” tests). Immunoassays can provide rapid, simple, and effective diagnoses for a variety of conditions. However, there remains a need in the art for improved immunoassays having increased sensitivity.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that the addition of detectable Fc-binding molecules (e.g., Protein A-conjugates, Protein G-conjugates, secondary antibody conjugates) can be used for the detection of antibodies in immunoassays, e.g., antigen-based capture or sandwich-type assays. These detectable Fc-binding molecules can be used either alone or in combination with other detectable entities for the detection of antibodies in immunoassays. For example, these Fc-binding molecules can be used to detect antibodies once the antibodies to be detected are captured by antibody-specific binding entities, e.g., antigens. In another example, these Fc-binding molecules can be used as a secondary source for detectable signals, e.g., in combination with another labeled or detectable antibody binding entity such as antigen.

This discovery can be applied to a variety of capture-type assays, related methods, compositions, and kits, as described herein.

Certain embodiments therefore include methods for detecting an antibody in a test sample comprising: (a) contacting the test sample with a first detector to form a first complex comprising the first detector and the antibody, wherein the first detector comprises an Fc-binding molecule conjugated to a first detectable entity; (b) contacting the first complex with a capture entity immobilized on a test region of a surface, wherein the capture entity is capable of specifically binding to the antibody; and (c) detecting the presence of a signal from the first detectable entity in the test region, wherein the presence of the signal is indicative of the presence of the antibody in the test sample. In certain embodiments, the first detector is immobilized to a conjugate region of the surface, wherein the conjugate region does not overlap with the test region of the surface. In specific embodiments, the Fc-binding molecule is protein A, protein G, or both. In certain embodiments, the first detector comprises protein A and protein G each conjugated to the detectable entity. In such embodiments, the ratio of protein A to protein G may be adjusted to optimize the level of signal amplification depending upon the type of immunoglobulin to be detected. For instance, in some embodiments, protein A and protein G are present in a ratio of about 10:1 to about 1:10, more preferably about 5:1 to about 1:5.

In certain embodiments, the capture entity is an antigen or antigenic peptide. In particular embodiments, said antigen or antigenic peptide is from an organism selected from the group consisting of heartworm, e.g., canine heartworm,Ehrlichia canis, Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Anaplasma phagocytophilum, feline leukemia virus, parvovirus, influenza A strain, influenza B strain, avian influenza virus, respiratory syncytial virus,Legionella, adenovirus, rotavirus, feline immunodeficiency virus, human immunodeficiency virus, and Group AStreptococcus.

In certain embodiments, the first detectable entity is a metal nanoparticle, metal nanoshell, fluorophore, or colored latex particle. In some embodiments, the metallic nanoparticle or metallic nanoshell is selected from the group consisting of gold particles, silver particles, copper particles, platinum particles, cadmium particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells.

Certain methods provided herein further comprise contacting the test sample with a second detector, wherein the second detector comprises an antigen or antigenic peptide conjugated to a second detectable entity, said antigen or antigenic peptide being capable of specifically binding to the antibody. In some embodiments, the first and second detector are immobilized on a conjugate region, wherein the conjugate region does not overlap with the test region of the surface. In certain embodiments, the conjugate region further comprises a control detector. The level of signal amplification can be selected by adjusting the ratio of the first detector to the second detector. In certain embodiments, the ratio of the first detector to the second detector is about 20:1 to about 1:20, more preferably about 20:1 to about 1:1.

In specific embodiments, the first and second detectable entities are the same. In more specific embodiments, the first and second detectable entities are both gold nanoparticles. In other embodiments, the first and second detectable entities are different.

In certain embodiments, the surface is a flow path in a lateral flow assay device, a surface of a microtiter plate or a flow path in an analytical rotor.

As noted above, certain embodiments employ a second detector that is conjugated to a detectable entity, where that second detector is an antigen or antigenic peptide. In some of these and related embodiments, the antigen or antigenic peptide is from an organism selected from the group consisting of heartworm, e.g., canine heartworm,Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Anaplasma phagocytophilum, Anaplasmaplatys, feline leukemia virus, parvovirus, e.g., canine parvovirus, influenza A strain, influenza B strain, avian influenza virus, respiratory syncytial virus,Legionella, adenovirus, rotavirus, feline immunodeficiency virus, human immunodeficiency virus, and Group AStreptococcus.

In some embodiments, the surface is a flow path in a lateral flow assay device or a flow path in an analytical rotor. In particular embodiments, the test sample is blood, serum, or plasma.

Also included are antibody detection devices, comprising a sample loading region; a conjugate region, wherein said conjugate region comprises a mobilizable first detector including an Fc-binding molecule conjugated to a first detectable entity; and a test region, wherein said test region comprises an immobilized capture entity capable of specifically binding to the antibody; wherein the sample loading region, the conjugate region and the test region are configured so that in operation a liquid sample when loaded into the sample loading region, is in fluid communication with the conjugate region and the test region. In certain embodiments, the Fc-binding molecule is protein A and/or protein G.

In some embodiments, the capture entity is an antigen or antigenic peptide. In particular embodiments, the antigen or antigenic peptide is from an organism selected from the group consisting of heartworm, e.g., canine heartwormEhrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Anaplasma phagocytophilum, Anaplasma platys, feline leukemia virus, parvovirus, e.g., canine parvovirus, influenza A strain, influenza B strain, avian influenza virus, respiratory syncytial virus,Legionella, adenovirus, rotavirus, feline immunodeficiency virus, human immunodeficiency virus, and Group AStreptococcus.

In particular embodiments, the first detectable entity is a metal nanoparticle, metal nanoshell, fluorophore, or colored latex particle. In specific embodiments, the metallic nanoparticle or metallic nanoshell is selected from the group consisting of gold particles, silver particles, copper particles, platinum particles, cadmium particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells.

In some embodiments, the device further comprises a control region in fluid communication with a liquid sample when it is loaded to the sample loading region. In certain embodiments, the control region comprises an immobilized binding partner capable of specifically binding a control detector.

In particular embodiments, the first detector comprises protein A or protein G conjugated to a first detectable entity and said immobilized binding partner is an anti-protein A or anti-protein G antibody.

Some devices further comprise an absorbent pad positioned downstream of the test region. In certain devices, the conjugate region is positioned upstream of the sample loading region. In some embodiments, the conjugate region is positioned downstream of the sample loading region. In some embodiments, the sample loading region comprises a blood separator material. In certain instances the conjugate region further comprises a mobilizable second detector, wherein the second detector comprises an antigen or antigenic peptide conjugated to a second detectable entity, said antigen or antigenic peptide being capable of specifically binding to the antibody. The ratio of the first detector (e.g. Fc-binding molecule, such as protein A and/or protein G, conjugated to a first detectable entity) to the second detector (antigen/antigenic peptide conjugated to a second detectable entity) can be adjusted to select a desired level of signal amplification. In some embodiments, the ratio of the first detector to the second detector is about 20:1 to about 1:20. In other embodiments, the ratio of the first detector to the second detector is about 20:1 to about 1:1.

In particular embodiments, the first and second detectable entities are the same. In specific embodiments, the first and second detectable entities are gold nanoparticles. In other embodiments, the first and second detectable entities are different.

In particular embodiments, for example, for detection of anti-microbial antibodies, the antigen or antigenic peptide is from an organism selected from the group consisting of heartworm, e.g., canine heartwormEhrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Anaplasma phagocytophilum, Anaplasma platys, feline leukemia virus, parvovirus, e.g., canine parvovirus, influenza A strain, influenza B strain, avian influenza virus, respiratory syncytial virus,Legionella, adenovirus, rotavirus, feline immunodeficiency virus, human immunodeficiency virus, and Group AStreptococcus.

Also included are kits comprising one or more of the detection devices and systems described herein, and instructions for using the device or system to detect an antibody in a test sample. Certain kits further comprise a second detector and instructions for combining the second detector with the test sample prior to application to the sample loading region of the detection system, wherein said second detector comprises an antigen or antigenic peptide conjugated to a second detectable entity, said antigen or antigenic peptide being capable of specifically binding to the antibody. In some embodiments, the instructions provide combining the second detector with the test sample such that the second detector will be present in a particular ratio with the first detector to achieve a desired level of signal amplification.

Also included are methods of detecting an antibody in a test sample comprising applying the test sample to the sample loading region of one or more of the detection devices or systems described herein, and detecting the presence or absence of a signal from the first detectable entity in the test region. Some methods further comprise combining a second detector with the test sample prior to application to the sample loading region of the detection system, wherein said second detector comprises an antigen or antigenic peptide conjugated to a second detectable entity, said antigen or antigenic peptide being capable of specifically binding to the antibody.

Certain embodiments relate to one or more capture complexes that comprise a capture entity, an antibody in a test sample, and a first detector, wherein the capture entity binds to the antibody and wherein the first detector comprises a Fc-binding molecule conjugated to a first detectable entity and binds to the Fc region of the antibody. Certain of these and related embodiments further comprise a second detector, wherein the second detector specifically binds to the variable region of the antibody. In some embodiments, the capture complex is immobilized on a test region of a surface.

