Enzyme Immunoassay with Fluorescence and Chemiluminescence Reaction

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This paper describes highly sensitive enzyme immunoassayes for insulin and cortisol using peroxidase as the label enzyme. The fluorescence reaction of homovanillic acid-H2O2 and chemiluminescence generating reaction of luminol-H2O2 with peroxidase are used as a means of determining peroxidase activity. The faint chemiluminescence is measured by a photon-counter (HTVC767). The peroxidase conjugate was synthesized according to NAKANE's method with slight modifications. The peroxidase-cortisol conjugate was also prepared by the mixed anhydride method. These conjugates were chromatographed on a Bio-Gel P-60 column. Various techniques for separating the bound and free labeled molecules were examined. Good results were obtained by the solid-phase method using anti-insulin or anti-cortisol-γ-globulin linked Sepharose 4B. The sensitivity of the assay, i.e., the amount of antigen displacing 10% of the initially bound tracer was fbund to be 0.2 to 0.5μU per tube and 25 to 30 pg of cortisol per tube. Upon testing a panel of 28 human plasma by the radioimmunoassay and the enzyme immunoassay (immuno-sorbent method) a coefficient of correlation between these two methods of r=0.916 was found. The recovery of 0.25 and 0.50ng of cortisol/ml was 85 and 83%, respectively.
Japan Society of Clinical Chemistryの論文