Simultaneous calibration heterogeneous immunoassay

In a method for determining the presence in a sample of an analyte, which analyte is a member of a specific binding pair ("mip") consisting of ligand and receptor ("antiligand"),

• said method employing a signal producing system having at least two enzymes, including one enzyme bound to a mip ("enzyme-bound-mip"), wherein a second ligand other than the ligand analyte is bonded to said enzyme, and a solute dye precursor which is catalytically transformed to an insoluble dye by one of said enzymes bound to a mip-containing measurement first surface, said insoluble dye producing a change in a detectable signal at said first surface in proportion to the amount of analyte, where said mip at said first surface provides for binding of enzyme-bound-mip through mip complex formation to said first surface, and contacting of said first surface with said sample and said enzyme-bound-mip results in binding of said enzyme-bound-mip to said first surface in proportion to the amount of analyte in said sample,
• the improvement which comprises:
• during said contacting, having adjacent to said first surface, a calibration second surface to which anti (second ligand) receptor is bound and to which enzyme of said enzyme-bound-mip becomes bound through mip complex formation in an amount which provides substantially predetermined ratios to the amount of enzyme-bound-mip bound to said first surface, whereby the ratio of the change in signal at said second surface to the change in signal at said first surface defines the amount of analyte in said sample substantially independent of non-specific factors.