Enzyme-linked immunosorbent assay (ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening.

We have developed a solid-phase ELISA for the specific and sensitive detect ion of apoptotic cells. This method is based on the ability of a monoclonal antibody (MAb) against single-stranded DNA (ssDNA) to specifically identif y apoptotic cells. The assay involves binding of cells to 96-well microtite r plates, treatment of the attached cells with formamide to denature DNA in apoptotic cells and one-step staining of the denatured DNA with a mixture of anti-ssDNA MAb and peroxidase-conjugated anti-mouse IgM. A near linear i ncrease in signal was seen as the number of apoptotic cells increased from 500 to 5000. Untreated and necrotic cells or cells with single-stranded DNA breaks induced by H2O2 did not produce signal above the background level. In leukemic cell cultures grown, treated with ID50 concentration of etoposi de, stained and analyzed in the same 96-well assay plate, intense ELISA sig nal was detected. The ratio of absorbance values from drug resistant and dr ug-sensitive cell lines treated with etoposide was in agreement with the de gree of resistance determined by growth inhibition assays. These data show that this ELISA has sufficient sensitivity for use in drug screening protoc ols. In breast cancer cell cultures treated with cisplatin, ELISA absorbanc e increased only after treatment with drug concentrations 10-fold higher th an concentrations inducing 95% growth inhibition. In cultures treated with staurosporine. there was a near linear relation between the ELISA absorbanc e values and cytotoxicity in the range of 15-92% growth inhibition. The abs ence of apoptotic signal in breast cancer cells treated with cytotoxic conc entrations of cisplatin indicated that this drug kills cells by non-apoptot ic mechanisms, whereas apoptosis was the dominant mechanism of cell death c aused by staurosporine. The formamide-MAb apoptosis ELISA described here ma y provide a basis for high-throughput screening of drugs based on their abi lity to induce or suppress apoptosis. (C) 2001 Elsevier Science B.V. All ri ghts reserved.