Assays for detecting analytes in samples and kits and compositions related thereto
The present invention provides methods of detecting analytes using particles having different physico-chemical properties, such as buoyancy, size, density, spectral characteristics, and/or binding properties, in solution-based sandwich assays and solution-based competition assays. The methods can be performed using rotors and bench-top centrifuges and provide for rapid, qualitative and quantitative detection of analytes. The present invention also provides kits that can be used to perform the methods, and mixtures containing particles suitable for the methods.
1. A method for detecting the presence of an analyte in a sample, wherein the analyte in the sample is free analyte, comprising:
mixing the sample with a population of first particles and a population of second particles to form a suspension, wherein the first particles comprise the analyte in bound form; and wherein the first particles and second particles are capable of forming multi-particle complexes,
removing multi-particle complexes from the suspension, and
detecting the presence of free first particles in the suspension after removal of the multi-particle complexes;
wherein an increase in the amount of free first particles in the suspension relative to the amount of free first particles present in a negative control suspension is indicative of the presence of the analyte in the sample.
2. The method of claim 1 , wherein the negative control suspension is prepared by a method comprising mixing a control sample that does not comprise the free analyte with the population of first particles and the population of second particles, and removing multi-particle complexes to form the negative control suspension.
3. The method of claim 1 , wherein the first particles comprise the analyte attached to or coating structures selected from the group consisting of colloidal nanoparticles, nanotubes, hollow nanospheres, and core-shell structures, wherein the structures comprise gold, silver, platinum, copper, or a composite of any of the foregoing metals.
4. The method of claim 1 , wherein the second particles comprise latex, polystyrene, polycarbonate, polyacrylate, PVDF, or silica.
5. The method of claim 1 , wherein the second particles comprise an antibody, antigen, polypeptide, polynucleotide, nucleoprotein, or aptamer.
6. The method of claim 5 , wherein the second particles comprise an antibody that recognizes an epitope on the analyte.
7. The method of claim 1 , wherein the free first particles remain in suspension at centrifugal forces of about 1600 g or less.
8. The method of claim 1 , wherein the first particles are smaller than the second particles.
9. The method of claim 1 , wherein the second particles sediment out of the suspension at centrifugal forces of about 1000 g to about 1600 g.
10. The method of claim 1 , wherein the first particles have a diameter of about 5 nm to about 40 nm and wherein the second particles have a diameter of about 400 nm to about 2000 nm.
11. The method of claim 1 , wherein the sample is in a liquid form and wherein the population of the first particles and population of second particles are in solid form prior to the mixing.
12. The method of claim 1 , wherein the analyte is an antigen selected from canine heartworm, feline leukemia virus, canine parvovirus, C-reactive protein, Giardia lamblia, Ehrlichia antigen or antibody, Borrelia antigen or antibody, and cardiac marker antigens.
13. The method of claim 1 , wherein removing the complex comprises using centrifugal force.
14. The method of claim 1 , wherein the population of first particles and population of second particles are separate prior to mixing with the sample.
15. The method of claim 1 , wherein the ratio of the average diameter of second particles to the average diameter of first particles is about 5:1 to about 50:1.