Immunoassay using an amplified cyclic detection system

Claim 1

In a method for determining a member of a ligand and receptor pair which comprises an assay wherein: (a) a sample of a first member of the ligand and receptor pair is contacted with a matrix bound second member of the pair whereby said first member becomes bound to said second member: (b) the thus produced matrix bound first member is contacted with a conjugate which binds specifically to the matrix bound first member which conjugate comprises a phosphatase whereby said phosphatase is bound to the matrix and (c) the matrix bound and unbound conjugate are separated and the matrix bound or the unbound conjugate is assayed; the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for NAD whereby NAD is produced, and (e) contacting the thus produced NAD with the components of a secondary system which is activated by the NAD, said secondary system comprising a cyclic chemical reaction which interconverts NAD and NADH, neither of which is present until the production of NAD by the conjugate, and which secondary system maintains the increase in concentration of NAD produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product at each turn of the cycle whereby amplification of the determinable change occurs.