Methods of reducing non-specific interaction in metal nanoparticle assays
The present invention discloses methods of reducing non-specific interactions of interfering species present in a sample in metallic nanoparticle-based assays, thereby increasing the sensitivity of these assays. In particular, the methods entail neutralizing the chemical reactivity of functional groups present in interfering species by addition of a neutralizing agent, such as an alkylating agent or heavy metal ion. The methods are especially useful in assays for the detection of analytes in biological samples. Reagent kits and assay mixtures for the practice of the described methods are also disclosed.
1. A method of reducing non-specific interaction of interfering species with a detectable reagent comprising:
mixing a neutralizing agent with the detectable reagent and a test sample,
wherein the detectable reagent is a binding partner coupled to a metallic nanoparticle or metallic nanoshell, wherein said binding partner specifically binds to an analyte in the test sample; and
wherein said neutralizing agent is an alkylating agent or a heavy metal ion that reduces the chemical reactivity of one or more interfering species present in the test sample.
2. The method of claim 1 , wherein said neutralizing agent reduces the reactivity of thiol, amino, imido, seleno, or carboxyl functional groups present in one or more interfering species.
3. The method of claim 1 , wherein said alkylating agent is selected from the group consisting of iodoacetamide, iodoacetate, N-ethylmaleimide, PEG-maleimide, ethlymethanesulfonate, and 4-vinylpyridine.
4. The method of claim 1 , wherein said heavy metal ion is selected from the group consisting of Hg (II), Ag (I), Bi (III), Cu (I), Cu (II), Cd (II), Zn (II), Pb (II), Pt (I), Pt (IV), Au (I), and Au (III).
5. The method of claim 1 , wherein one or more interfering species comprises one or more macromolecules that exhibit a non-specific binding affinity to the detectable reagent.
6. The method of claim 1 , wherein said test sample is a biological sample.
7. The method of claim 1 , wherein said binding partner is a macromolecule.
8. The method of claim 1 , wherein said metallic nanoparticle or metallic nanoshell is selected from the group consisting of gold particles, silver particles, copper particles, platinum particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells.
9. The method of claim 7 , wherein said binding partner is an antibody.
10. The method of claim 9 , wherein said antibody binds an antigen selected from the group consisting of epitopes of heartworm, canine pancreatic lipase, 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, and Group A Streptococcus.
11. The method of claim 7 , wherein said macromolecule is a nucleic acid, a polynucleotide, an antibody, an antigen, a receptor, a ligand, a protein, or a peptide.
12. The method of claim 1 , wherein said analyte is an antigen selected from the group consisting of epitopes of heartworm, canine pancreatic lipase, 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, and Group A Streptococcus.
13. A reagent kit comprising:
an isolated binding partner coupled to a metallic nanoparticle or metallic nanoshell, wherein said isolated binding partner specifically binds to an analyte in a biological sample; and
a neutralizing agent, wherein said neutralizing agent is an alkylating agent or a heavy metal ion,
wherein said isolated binding partner and said neutralizing agent are present in the same formulation.
14. The reagent kit of claim 13 , wherein said alkylating agent is selected from the group consisting of iodoacetamide, iodoacetate, N-ethylmaleimide, PEG-maleimide, ethlymethanesulfonate, and 4-vinylpyridine.
15. The reagent kit of claim 13 , wherein said heavy metal ion is selected from the group consisting of Hg (II), Ag (I), Bi (III), Cu (I), Cu (II), Cd (II), Zn (II), Pb (II), Pt (I), Pt (IV), Au (I), and Au (III).
16. The reagent kit of claim 13 , further comprising a blocking agent selected from the group consisting of bovine serum albumin, casein, gelatin, ovalbumin, and gamma-globulins.
17. The reagent kit of claim 16 , further comprising a sugar.
18. The reagent kit of claim 17 , further comprising a detergent.
19. The reagent kit of claim 13 , wherein the formulation is a dry powder.
20. The reagent kit of claim 13 , wherein the formulation is lyophilized.
21. The reagent kit of claim 20 , wherein the formulation is in the form of a pellet or a bead.
22. The reagent kit of claim 13 , wherein said metallic nanoparticle or metallic nanoshell is selected from the group consisting of gold particles, silver particles, copper particles, platinum particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells.
23. The reagent kit of claim 13 , wherein said isolated binding partner coupled to a metallic nanoparticle or metallic nanoshell is an antibody that specifically binds to an analyte in a biological sample.
24. The reagent kit of claim 23 , wherein said antibody binds to an analyte selected from the group consisting of epitopes of heartworm, canine pancreatic lipase, 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, and Group A Streptococcus.
25. An assay mixture comprising
a biological sample;
a neutralizing agent, wherein said neutralizing agent is an alkylating agent or a heavy metal ion; and
a detectable reagent, wherein said detectable reagent is a binding partner coupled to a metallic nanoparticle or metallic nanoshell, wherein said binding partner specifically binds to an analyte in the biological sample.
26. The assay mixture of claim 25 , wherein said alkylating agent is selected from the group consisting of iodoacetamide, iodoacetate, N-ethylmaleimide, PEG-maleimide, ethlymethanesulfonate, and 4-vinylpyridine.
27. The assay mixture of claim 25 , wherein said heavy metal ion is selected from the group consisting of Hg (II), Ag (I), Bi (III), Cu (I), Cu (II), Cd (II), Zn (II), Pb (II), Pt (I), Pt (IV), Au (I), and Au (III).
28. The assay mixture of claim 25 , wherein said binding partner is an antibody.
29. The reagent kit of claim 13 , wherein said isolated binding partner is a macromolecule.
30. The reagent kit of claim 29 , wherein said macromolecule is a nucleic acid, a polynucleotide, an antibody, an antigen, a receptor, a ligand, a protein, or a peptide.
31. The assay mixture of claim 25 , wherein said neutralizing agent is present in a concentration of about 0.5 mM to about 50 mM.
32. The assay mixture of claim 25 , wherein said neutralizing agent is present in a concentration of about 1 mM to about 10 mM.
33. The assay mixture of claim 25 , wherein said binding partner is a macromolecule.
34. The assay mixture of claim 33 , wherein said macromolecule is a nucleic acid, a polynucleotide, an antibody, an antigen, a receptor, a ligand, a protein, or a peptide.
35. The reagent kit of claim 13 , further comprising instructions for mixing the isolated binding partner and the neutralizing agent with a biological sample to minimize non-specific interactions in a detection assay.
36. The assay mixture of claim 25 , wherein said metallic nanoparticle or metallic nanoshell is selected from the group consisting of gold particles, silver particles, copper particles, platinum particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells.
37. The assay mixture of claim 25 , further comprising polyethylene glycol.