Novel assays for detecting analytes in samples and kits and compositions related thereto

Abstract

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 of detecting an analyte in a sample comprising
 mixing a sample with a population of first particles and a population of second particles to form a suspension, wherein the first and second particles are different and capable of forming a multi-particle complex comprising the first particle, second particle, and the analyte,   removing multi-particle complexes from the suspension, and   detecting the presence of first particles in the suspension,   wherein a decrease of the first particles in the suspension is indicative of the presence of the analyte in the sample.   
     
     
         2 . The method of  claim 1 , wherein the first particles or second particles comprise an antibody, antigen, polypeptide, polynucleotide, nucleoprotein, or aptamer. 
     
     
         3 . The method of  claim 1 , wherein the first particles comprise a first monoclonal antibody or a first polyclonal antibody recognizing a first epitope on the analyte, wherein the second particles comprise a second monoclonal antibody or a second polyclonal antibody recognizing a second epitope on the analyte, and wherein the first epitope does not overlap with the second epitope. 
     
     
         4 . The method of  claim 1 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise gold, silver, platinum, copper, or a composite of any of the foregoing metals. 
     
     
         5 . The method of  claim 1 , wherein free first particles remain in suspension at centrifugal forces of about 1600 g or less. 
     
     
         6 . The method of  claim 1 , wherein the first particles are smaller than the second particles. 
     
     
         7 . The method of  claim 1 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise gold, silver, platinum, copper, or a composite of any of the foregoing metals, and wherein the second particles comprise latex, polystyrene, polycarbonate, polyvinylidene fluoride (PVDF), or silica. 
     
     
         8 . The method of  claim 1 , wherein free first particles remain in suspension at centrifugal forces of about 1600 g or less, and wherein free second particles sediment out of the suspension at centrifugal forces of about 1000 g to about 1600 g. 
     
     
         9 . The method of  claim 1 , wherein the first particles have an average diameter of about 5 nm to about 40 nm, and wherein the second particles have an average diameter of about 400 nm to about 2000 nm. 
     
     
         10 . The method of  claim 1 , wherein the sample is in a liquid form and wherein the population of first particles and the population of second particles are in solid form prior to the mixing. 
     
     
         11 . The method of  claim 1 , wherein the analyte is canine heartworm, feline leukemia virus, canine parvovirus, C-reactive protein,  Giardia lamblia, Ehrlichia  antigen or antibody,  Borrelia  antigen or antibody, and cardiac marker antigens. 
     
     
         12 . The method of  claim 1 , wherein removing the multi-particle complexes comprises using centrifugal force. 
     
     
         13 . The method of  claim 1 , wherein the population of first particles and the population of second particles are separate prior to mixing with the sample. 
     
     
         14 . 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. 
     
     
         15 . A method for detecting an analyte in a sample comprising
 mixing a sample with a population of first particles and a population of second particles to form a suspension, wherein the first particle comprises the analyte, and wherein the first particle and second particle are capable of forming multi-particle complexes,   removing multi-particle complexes from the suspension, and   detecting the presence of the first particles in the suspension,   wherein a increase of the first particles in the suspension is indicative of the presence of the analyte in the sample.   
     
     
         16 . The method of  claim 15 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise gold, silver, platinum, copper, or a composite of any of the foregoing metals. 
     
     
         17 . The method of  claim 15 , wherein the second particles comprise latex, polystyrene, polycarbonate, polyacrylate, PVDF, or silica. 
     
     
         18 . The method of  claim 15 , wherein free first particles remain in suspension at centrifugal forces of about 1600 g or less. 
     
     
         19 . The method of  claim 15 , wherein the first particles are smaller than the second particles. 
     
     
         20 . The method of  claim 15 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise gold, silver, platinum, copper, or a composite of any of the foregoing metals, and wherein the second particles comprise latex, polystyrene, polycarbonate, polyvinylidene fluoride (PVDF), or silica. 
     
     
         21 . The method of  claim 15 , wherein free first particles remain in suspension at centrifugal forces of about 1600 g or less, and wherein free second particles sediment out of the suspension at centrifugal forces of about 1000 g to about 1600 g. 
     
     
         22 . The method of  claim 15 , wherein the first particle has a diameter of about 5 nm to about 40 nm and wherein the second particle has a diameter of about 400 nm to about 2000 nm. 
     
     
         23 . The method of  claim 15 , 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. 
     
     
         24 . The method of  claim 15 , wherein the analyte is canine heartworm, feline leukemia virus, canine parvovirus, C-reactive protein,  Giardia lamblia, Ehrlichia  antigen or antibody,  Borrelia  antigen or antibody, and cardiac marker antigens. 
     
     
         25 . The method of  claim 15 , wherein removing the complex comprises using centrifugal force. 
     
     
         26 . The method of  claim 15 , wherein the population of first particles and population of second particles are separate prior to mixing with the sample. 
     
     
         27 . The method of  claim 15 , 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. 
     
     
         28 . A kit comprising a population of first particles and a population of second particles, wherein the first particles and second particles are different and capable of forming a complex comprising the first particle, second particle and an analyte, and 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. 
     
     
         29 . The kit of  claim 28 , wherein the population of the first particles and the population of the second particles are unmixed. 
     
     
         30 . The kit of  claim 28 , wherein the population of the first particles and the population of the second particles are in solid form. 
     
     
         31 . The kit of  claim 28 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise gold, silver, platinum, copper, or a composite of any of the foregoing metals, and wherein the second particles comprise latex, polystyrene, polycarbonate, polyvinylidene fluoride (PVDF), or silica. 
     
     
         32 . The kit of  claim 28 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise (i) gold, silver, platinum, or copper and (ii) an analyte, and wherein the second particles comprise (i) latex, polystyrene, polycarbonate, polyvinylidene fluoride (PVDF), or silica and (ii) an antibody capable of recognizing an epitope of the analyte. 
     
     
         33 . The kit of  claim 28 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise (i) gold, silver, platinum, or copper and (ii) an antibody capable of recognizing a first epitope on an analyte, wherein the second particles comprise (i) latex, polystyrene, polycarbonate, polyvinylidene fluoride (PVDF), or silica and (ii) an antibody capable of recognizing a second epitope on the same analyte, and wherein the first and second epitopes are non-overlapping. 
     
     
         34 . A mixture comprising an analyte, a population of first particles, a population of second particles, and a population of multi-particle complexes comprising a first particle, a second particle, and an analyte, 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. 
     
     
         35 . The mixture of  claim 34 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise gold, silver, platinum, or copper, and wherein the second particles comprise latex, polystyrene, polycarbonate, polyvinylidene fluoride (PVDF), or silica. 
     
     
         36 . The mixture of  claim 34 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise (i) gold, silver, platinum, or copper and (ii) an analyte, and wherein the second particles comprise (i) latex, polystyrene, polycarbonate, polyvinylidene fluoride (PVDF), or silica and (ii) an antibody capable of recognizing an epitope of the analyte 
     
     
         37 . The mixture of  claim 34 , wherein the first particles are colloidal nanoparticles, nanotubes, hollow nanospheres, or core-shell structures that comprise (i) gold, silver, platinum, or copper and (ii) an antibody capable of recognizing a first epitope on an analyte, wherein the second particles comprise (i) latex, polystyrene, polycarbonate, polyvinylidene fluoride (PVDF), or silica and (ii) an antibody capable of recognizing a second epitope on the same analyte, and wherein the first and second epitopes are non-overlapping.