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 detection of apoptotic cells. This method is based on the ability of a monoclonal antibody (MAb) against single-stranded DNA (ssDNA) to specifically identify apoptotic cells. The assay involves binding of cells to 96-well microtiter 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 increase 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 H(2)O(2) did not produce signal above the background level. In leukemic cell cultures grown, treated with ID(50) concentration of etoposide, stained and analyzed in the same 96-well assay plate, intense ELISA signal was detected. The ratio of absorbance values from drug resistant and drug-sensitive cell lines treated with etoposide was in agreement with the degree of resistance determined by growth inhibition assays. These data show that this ELISA has sufficient sensitivity for use in drug screening protocols. In breast cancer cell cultures treated with cisplatin, ELISA absorbance increased only after treatment with drug concentrations 10-fold higher than concentrations inducing 95% growth inhibition. In cultures treated with staurosporine, there was a near linear relation between the ELISA absorbance values and cytotoxicity in the range of 15-92% growth inhibition. The absence of apoptotic signal in breast cancer cells treated with cytotoxic concentrations of cisplatin indicated that this drug kills cells by non-apoptotic mechanisms, whereas apoptosis was the dominant mechanism of cell death caused by staurosporine. The formamide-MAb apoptosis ELISA described here may provide a basis for high-throughput screening of drugs based on their ability to induce or suppress apoptosis.
Frankfurt
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Experimental Therapeutics Division, Radiation Oncology Department, University of Miami Medical School, Sylvester Cancer Center, 33136, Miami, FL, USA. apostain@bellsouth.net
Krishan
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A
D000911:Antibodies, Monoclonal / Q000276:immunology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D002455:Cell Division; D004277:DNA, Single-Stranded / Q000276:immunology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005559:Formamides / Q000737:chemistry; D006801:Humans; D009369:Neoplasms / Q000473:pathology; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D004277:DNA, Single-Stranded; D005559:Formamides; C031066:formamide