Signal amplification in lateral flow and related immunoassays
The present invention provides methods, devices, compositions (e.g., capture complexes), and kits useful for enhancing the detection of antibodies in a test sample. The methods, devices, and compositions utilize detectable Fc-binding molecules such as Protein A, Protein G, and/or an Fc-specific antibody to amplify the signal of a detected antibody in immunoassays, such as lateral flow assays.
1. An antibody detection device comprising:
a sample loading region;
a conjugate region, wherein said conjugate region comprises (i) a mobilizable first detector comprising an Fc-binding molecule capable of binding to the Fc region of the antibody, wherein the Fc-binding molecule is conjugated to a first detectable entity, and (ii) a mobilizable second detector comprising an antigen or antigenic peptide conjugated to a second detectable entity, said antigen or antigenic peptide being capable of specifically binding to the variable region of the antibody; and
a test region, wherein said test region comprises an immobilized capture entity capable of specifically binding to the variable region of the antibody;
wherein the sample loading region, the conjugate region and the test region are configured so that in operation a liquid sample comprising the antibody, when loaded into the sample loading region, is in fluid communication with the conjugate region and the test region.
2. The detection device of claim 1 , wherein the Fc-binding molecule is protein A and/or protein G.
3. The detection device of claim 1 , wherein the capture entity is an antigen or antigenic peptide.
4. The detection device of claim 3 , wherein said antigen or antigenic peptide is from an organism selected from the group consisting of heartworm, Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Anaplasma phagocytophilum, Anaplasma platys , 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 A Streptococcus.
5. The detection device of claim 1 , wherein the first detectable entity is a metal nanoparticle, metal nanoshell, fluorophore, or colored latex particle.
6. The detection device of claim 5 , wherein 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.
7. The detection device of claim 1 , wherein said device further comprises a control region in fluid communication with a liquid sample when it is loaded to the sample loading region.
8. The detection device of claim 7 , wherein said control region comprises an immobilized binding partner capable of specifically binding a control detector.
9. The detection device of claim 8 , wherein said 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.
10. The detection device of claim 1 , further comprising an absorbent pad positioned downstream of the test region.
11. The detection device of claim 1 , wherein said conjugate region is positioned upstream of said sample loading region.
12. The detection device of claim 1 , wherein said conjugate region is positioned downstream of said sample loading region.
13. The detection device of claim 1 , wherein said sample loading region comprises a blood separator material.
14. The detection device of claim 1 , wherein the first and second detectable entities are the same type of detectable entity.
15. The detection device of claim 14 , wherein the first and second detectable entities are gold nanoparticles.
16. The detection device of claim 1 , wherein the first and second detectable entities are different types of detectable entities.
17. The detection device of claim 1 , wherein said antigen or antigenic peptide is from an organism selected from the group consisting of heartworm, Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Anaplasma phagocytophilum, Anaplasma platys , 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 A Streptococcus.
18. The detection device of claim 1 , wherein the first mobilizable detector and the second mobilizable detector are present in a ratio of about 20:1 to about 1:1.
19. The detection device of claim 18 , wherein the first mobilizable detector comprises an Fc-binding molecule conjugated to a first detectable entity, and wherein the Fc-binding molecule is protein A and/or protein G.
20. The detection device of claim 18 , wherein the first mobilizable detector comprises protein A and protein G each conjugated to a first detectable entity.
21. The detection device of claim 20 , wherein protein A and protein G are present in a ratio of about 10:1 to about 1:10.
22. A kit comprising the detection device of claim 1 and instructions for using the device to detect an antibody in a test sample.
23. The kit of claim 22 , further comprising instructions for combining the second detector with the test sample prior to application to the sample loading region of the detection device.
24. The kit of claim 22 , wherein the first mobilizable detector and the second detector are present in a ratio of about 20:1 to about 1:1.
25. The kit of claim 24 , wherein the first mobilizable detector comprises an Fc-binding molecule conjugated to a first detectable entity, and wherein the Fc-binding molecule is protein A and/or protein G.
26. The kit of claim 24 , wherein the first detector comprises protein A and protein G each conjugated to a first detectable entity.
27. The kit of claim 26 , wherein protein A and protein G are present in a ratio of about 10:1 to about 1:10.
28. The kit of claim 22 , further comprising instructions for combining the first detector, the second detector, and the test sample together in a mixture prior to application of the mixture to the sample loading region of the detection device.