Determination of one thousandth of an attomole (1 zeptomole) of alkaline phosphatase: application in an immunoassay of proinsulin.

Enzyme amplification has proved to be a highly sensitive quantification technique for immunoassays. We have shown that by using a fluorescent end-point, even more sensitive enzyme amplification assays can be generated than hitherto reported. We describe some general properties of this system and demonstrate its application in an assay for human proinsulin in plasma. The detection system can be used to measure less than one thousandth of an attomole (1 zeptomole) of alkaline phosphatase, equivalent to about 350 molecules of alkaline phosphatase per well of a microtiter plate. We have used this system to construct a proinsulin assay with a sensitivity of 0.017 pmol/L.