AMPLIFICATION OF ENZYME SIGNAL IN ENZYME - LINKED IMMUNOSORBENT-ASSAY OF HUMAN ALPHA-FETOPROTEIN

Enzyme linked immunosorbent assay (ELISA) is now used for the quantitation of a wide range of clinically important substances and have, in many cases, replaced radioimmunoassay though without improving its sensitivity. In this paper, the potential of enzyme amplification system for greatly enhancing both the sensitivity and the speed of ELISA is described. The principle of the new approach is that alkaline phosphatase was used as the labelling enzyme to catalyze the dephosphorylation of nicotinamide adenine dinucleotide phosphate (NADP+). The nicotinamide adenine dinucleotide (NAD+) formed catalytically activated a strictly NAD+ -specific redox cycle which produced an intensely colored formazan dye as the end product. Each molecule of product from the first reaction took part in many cycles of the second reaction, thus amplifying the signal. As a result, the enzyme amplification increased the absorbance obtained from the commonly used enzyme label alkaline phosphatase by at least 250 fold. The application of this approach to ELISA made possible an assay for human alphafetoprotein (hAFP) which was rapid, precise and 40 times more sensitive than the conventional ELISA using p-nitrophenyl phosphate as the enzyme substrate. Moreover, the present method gave good correlation with the RIA method. Thus it is appropriate for routine assaying minute quantities of hAFP in any clinical specimens.