Measurement of antibody-dependent cell-mediated cytotoxicity by Hb-enzyme release assay (Hb-ERA).

A non-isotope method, hemoglobin-enzyme release assay (Hb-ERA), for measuring antibody-dependent cell-mediated cytotoxicity (ADCC) has been successfully developed. The assay is based on the fact that hemoglobin has peroxidase activity capable of catalyzing the oxidation of o-phenylenediamine to give coloration which can be measured by spectrophotometer. In comparison to the conventional 51Cr release assay, Hb-ERA is economic, sensitive, reproducible and does not have the shortcomings associated with manipulation of radioactive substances. It is applicable in clinical and research laboratories of any scale. The optimum conditions for the assay are as follows: an effector cell: target cell ratio of 10:1 and an incubation period of 14-16 h at 37 degrees C in 5% CO2. The splenic ADCC activity of mice was measured. The cytotoxic index of mice aged 10-14 weeks (39.71 +/- 11.19) was significantly higher than that of 20-24 week-old mice (18.42 +/- 10.31) (P less than 0.001). The ADCC activity is not influenced by sex. The merits and drawbacks of Hb-ERA are objectively evaluated.