Enzyme inhibitory homogeneous immunoassay for high molecular weight antigen (I)

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Abstract
A highly sensitive homogeneous enzyme immunoassay has been developed for the determination of antigens with high molecular weight. The method presented is based on steric hindrance of the enzyme against an insoluble substrate by antigen binding to antibody: enzyme, conjugate, and consists of α‐amylase from Bacillus subtilis as label with antibody, human α‐fetoprotein (AFP) or human ferritin as ligands, and insoluble substrate. The assay procedure is as follows: sample is mixed and incubated with the conjugate at 37° C for 20 min. Thereafter, the mixture is incubated at 37° C for 20 min following addition of substrate, and the enzyme reaction is terminated by adding the stopper. Then the mixture is centrifuged at 3,000 rpm for 1 minute, and the absorbance of the supernatant is measured at 620 nm. The measurable range for AFP was 5‐200 ng/ml for ferritin, it was 10‐800 ng/ml. In addition, the method was well correlated with radioimmunoassay; i.e., for AFP r = 0.892 and for ferritin r = 0.951. Furthermore, there was no interference during the assay of endogenous a‐amylase in human serum as a result of the use of the inhibitor specific to mammalian amylase which was purified from bacteria.