A SEQUENTIAL BINDING ASSAY WITH A WORKING RANGE EXTENDING BEYOND 7 ORDERS OF MAGNITUDE

A new immunometric sequential binding assay has been developed in whic h the sample is first reacted with a solid phase binding partner in lo w concentration, and subsequently with a second binding partner at a h igher concentration. The amounts of analyte bound to the two solid pha se binding partners are separately measured, thus establishing a doubl e standard curve. There is a shift between the two standard curves alo ng the concentration axis. Thus an unambiguous determination of analyt e concentration is obtained, even in the descending region of the curv es where the 'hook' effect causes decreasing signal with increasing an alyte concentration. A two-particle immunofluorometric assay for AFP b ased on this principle measured by flow cytometry, resulted in an assa y with rapid binding (similar to 2 h), a detection limit of 0.1 kIU/l and a working range (0.3 to > 3 x 10(6) kIU/l) in excess of 7 log(10) orders. Assay results compared well with those of an immunoradiometric assay.