A microfluorometric mithramycin assay for quantitating the effects of immunotoxicants on lymphocyte activation

A semiautomated, microfluorometric assay has been developed for the detection of toxicant-induced changes in lymphocyte DNA content at standard intervals after mitogen activation. DNA is quantitated by solubilizing the cells and determining the fluorescence enhancement that results from formation of the highly specific mithramycin:DNA adduct. The limit of detection is 0.21 microgram (30,000 resting cell equivalents) per microtiter well. Correlation with the less sensitive, nonautomatable, diphenylamine DNA assay gives a correlation coefficient r = 0.91. Prototype substances representative of true immunotoxicants (prostaglandin E2) and common interfering substances (thymidine at less than 5 X 10(-4) M)m have been tested. The latter substance produces false positive results in the standard [3H]thymidine assay. The mithramycin assay does not inappropriately detect this interfering substance. It has the characteristics of a highly specific, accurate technique for screening and quantitating immunotoxic drugs, agents, and mediators in patient sera and other complex biological fluids.