Assay with increased dynamic range
Provided herein are assays and kits useful for avoiding “prozone phenomenon” or “hook effect” and which expand the range of accurately measurable analyte concentrations.
1. A method of expanding the dynamic range of an assay, comprising:
a) contacting a test sample suspected of comprising an analyte with a tracer comprising said analyte or fragment thereof attached to a label, a first analyte-binding molecule attached to a first solid support, a second analyte-binding molecule attached to a second solid support, wherein the binding affinity for the analyte of the first analyte-binding molecule is greater than that of the second analyte-binding molecule, wherein the first analyte-binding molecule and the second analyte-binding molecule do not concurrently bind to the analyte;
b) measuring the signal intensities from the tracer bound to the first analyte-binding molecule on the first solid support and the second analyte-binding molecule on the second solid support; and
c) establishing a flag value at or near the leveling off value (plateau) of the signal intensity of the tracer bound to the first analyte-binding protein attached to the first solid support wherein the flag value is a threshold or cut-off value that governs whether the signal from the analyte-binding molecule with relatively higher binding affinity for the analyte is used in determining the concentration of analyte in a test sample.
2. The method of claim 1 , wherein when the signal intensity of the tracer bound to the first analyte-binding protein attached to the first solid support is equal to or less than the flag value, then the signal intensity of the tracer bound to the second analyte-binding protein attached to the second solid support is used to determine analyte concentration.
3. The method of claim 1 , wherein one or more of the first analyte-binding molecule and/or the second analyte-binding molecule is an antibody or fragment thereof.
4. The method of claim 1 , wherein the label is selected from the group consisting of an enzyme, a chromophore, and a fluorophore.
5. The method of claim 1 , wherein the first solid support and the second solid support are independently selected from the group consisting of a particle, a microparticle, a bead, an electrode and a multiwell plate.
6. The method of claim 1 , wherein the first solid support is a microparticle or bead comprising a first chromophore and the second solid support is a microparticle or bead comprising a second chromophore.
7. The method of claim 1 , wherein the first solid support and the second solid support are microparticles which differ in either shape or size.
8. The method of claim 1 , wherein the first solid support and the second solid support are different.
9. The method of claim 1 , wherein the first solid support is a first electrode and the second solid support is a second electrode, wherein the first electrode and the second electrode are spatially separated.
10. The method of claim 1 , wherein the difference in binding affinity of the first analyte-binding molecule and the second analyte-binding molecule for the analyte ranges from about 5-fold to about 100-fold.
11. The method of claim 1 , wherein the difference in binding affinity of the first analyte-binding molecule and the second analyte-binding molecule for the analyte is at least about 100-fold.