New method for nitroblue-tetrazolium (NBT) test was developed by the following modifications. (1) After 10 N KOH treatment, bathochromic shift in the maximal absorption took place from 515 nm to 710 nm and the optical density of the new peak was 5 times higher than that of non-treated sample. In this assay, dimethylformamide extraction was substituted for pyridine. (2) Because of the smallness of the incubation mixture (100 μl), the background of the test was greatly reduced. (3) Increase in the concentration of latex particles elevated the NBT reducing activity. In consideration with the background absorption, 2.9-5.7×108 particles/assay was set as the optimal condition. The particles themselves, however, did not interrupt the reading of optical density in this system. In this modified method, 3 ml of blood was enough to estimate the NBT reducing activity in the duplicate tests (6 samples). This assay system is, therefore, applicable for weak children, from whom the collection of the large volume of the blood is not desirable.
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Okamura et al. (1976) studied this question.