Multiplexed assay methods

1. A method for measuring analytes in a sample, the method comprising:

a) contacting a first dilution of said sample with one or more binding surfaces having binding reagents that bind a first analyte and a second analyte, wherein said one or more binding surfaces are located within a measurement chamber;

b) contacting said binding surface(s) with a first detection reagent, wherein said first detection reagent binds to said first analyte;

c) contacting a second dilution of said sample with said binding surface(s) in said measurement chamber, said second dilution having a dilution factor less than said first dilution;

d) contacting said binding surface(s) with a second detection reagent, wherein said second detection reagent binds to said second analyte; and

e) measuring the amount of said first detection reagent and said second detection reagent bound to said binding surface(s).

2. A method for measuring analytes in a sample, the method comprising:

a) contacting a first dilution of said sample with one or more binding surfaces in a measurement chamber;

b) contacting said binding surface(s) in said measurement chamber with a first detection reagent;

c) contacting a second dilution of said sample with said binding surface(s) in said measurement chamber, said second dilution having a dilution factor less than said first dilution;

d) contacting said binding surface(s) in said measurement chamber with a second detection reagent; and

e) measuring the amount of said first detection reagent and said second detection reagent bound to said binding surface(s)

wherein

i) said binding surface(s) and said first detection reagent bind said first analyte;

ii) said binding surface(s) binds said first analyte and said first detection reagent competes with said first analyte for said binding surface(s);

iii) said first binding reagent binds said first analyte and said binding surface(s) competes with said first analyte for binding to said first detection reagent;

and wherein

i) said binding surface(s) and said second detection reagent bind said second analyte;

ii) said binding surface(s) binds said second analyte and said second detection reagent competes with said second analyte for said binding surface(s);

iii) said second binding reagent binds said second analyte and said binding surface(s) competes with said second analyte for binding to said second detection reagent.

3. The method of claims 1 or 2 , wherein said one or more binding surfaces comprises two discrete binding domains, a first binding domain comprising a first binding reagent that binds said first analyte and a second binding domain comprising a second binding reagent that binds said second analyte.

4. The method of claims 1 or 2 , wherein the concentration of said first analyte in said sample is higher than the concentration of said second analyte.

5. The method of claims 1 or 2 , wherein said sample is diluted in said first dilution by a factor of 10.

6. The method of claims 1 or 2 further comprising washing said first detection reagent from said binding surface(s) prior to contacting said binding surface(s) with said second dilution of said sample.

7. The method of claims 1 or 2 further comprising blocking unbound sites for said first analyte on said binding surface(s) after contacting said binding surface(s) with said first dilution of said sample but prior to contacting said binding surface(s) with said second dilution of said sample.

8. The method of claim 2 , wherein said binding surface(s) has at least one binding reagent immobilized thereon.

9. The method of claim 8 , wherein said binding reagent binds said analyte of interest.

10. The method of claim 9 , further comprising contacting said sample with a bridging reagent that binds said binding reagent and said analyte.

11. The method of claims 1 or 2 , wherein said binding surface(s) is an electrode surface(s).

12. The method of claims 1 or 2 , wherein said binding surface(s) is an electrode surface(s) on the bottom of a multi-well plate.

13. The method of claims 1 or 2 , wherein said binding surface(s) is a surface of a particle(s).

14. The method of claims 1 or 2 , wherein said binding surface(s) is a surface of a magnetic particle(s).

15. The method of claims 1 or 2 , wherein said binding surface(s) is a surface in a well(s) of a multi-well ELISA plate.

16. The method of claims 1 or 2 , wherein said binding surface(s) is a surface of a glass slide.

17. The method of claims 1 or 2 , wherein said first and second detection reagents are labeled with a label.

18. The method of claims 17 , wherein said first detection reagent is labeled with a first label and said second detection reagent is labeled with a second label, and said first label and said second label are measurably distinct.

19. The assay of claim 18 , wherein said label is selected from a group comprising ECL label, luminescent label, fluorescent label, phosphorescent label, radioactive label or light scattering label.

20. The method of claims 1 or 2 , wherein said amounts of said first and second detection reagents are measured simultaneously.