Particle Analysis Assay for Biomolecular Quantification

Abstract

A method is provided for carrying out multi-step separations of objects bearing at least two binding sites. In the first step, a first binder/bead composition is bound to objects that bear the first binding site, and then unbound objects, i.e. objects not bearing the first binding site, are separated from bound objects. In the second step, a second binder/bead composition is bound to the remaining objects that bear the second binding site, and then the objects that are bound to both beads are removed from those objects that are bound to only one bead. The beads can differ in magnetic responsiveness, charge, size, color, and the like, and these differences can be used to carry out the separation steps.

1 .- 20 . (canceled) 
     
     
         21 . A method of quantifying a desired protein in a sample comprising the steps of:
 (a) obtaining a solid support coated with a first antibody that recognizes the desired protein;   (b) contacting the antibody-coated solid support with the sample to allow the first antibody to bind to the desired protein;   (c) attaching a second antibody to the desired protein, wherein the second antibody comprises a modification such that the second antibody may be attached to a first set of microbeads;   (d) attaching first set of microbeads to the second antibody;   (e) washing to remove microbeads that are not attached to the second antibody; and   (f) counting the microbeads that are attached to the second antibody to determine the quantity of the desired protein; and wherein steps (a)-(d) may be performed in any order or simultaneously.   
     
     
         22 . The method of  claim 21 , wherein the solid support comprises a second set of microbeads that is distinguishable from the first set of microbeads by size, charge, color, or attachability to a solid support. 
     
     
         23 . The method of  claim 21 , wherein the modification comprises a biotin molecule and the fist set of microbeads comprise streptavidin. 
     
     
         24 . The method of  claim 21 , further comprising the step of releasing the microbeads attached to the second antibody by protease treatment prior to the counting step.