Frankfurt O S, Krishan A
Experimental Therapeutics Division, Radiation Oncology Department, University of Miami Medical School, Sylvester Cancer Center, 33136, Miami, FL, USA.
J Immunol Methods. 2001 Jul 1;253(1-2):133-44. doi: 10.1016/s0022-1759(01)00387-8.
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.
我们开发了一种用于特异性和灵敏检测凋亡细胞的固相酶联免疫吸附测定(ELISA)。该方法基于一种抗单链DNA(ssDNA)的单克隆抗体(MAb)特异性识别凋亡细胞的能力。该检测方法包括将细胞结合到96孔微量滴定板上,用甲酰胺处理贴壁细胞以使凋亡细胞中的DNA变性,并用抗ssDNA MAb和过氧化物酶偶联的抗小鼠IgM混合物对变性DNA进行一步染色。随着凋亡细胞数量从500增加到5000,信号呈近似线性增加。未经处理的坏死细胞或由H(2)O(2)诱导产生单链DNA断裂的细胞未产生高于背景水平的信号。在白血病细胞培养物中,用依托泊苷的半数抑制浓度(ID(50))处理,在同一96孔检测板中进行染色和分析,检测到强烈的ELISA信号。用依托泊苷处理的耐药和敏感细胞系的吸光度值之比与通过生长抑制试验确定的耐药程度一致。这些数据表明,这种ELISA在药物筛选方案中具有足够的灵敏度。在用顺铂处理的乳腺癌细胞培养物中,ELISA吸光度仅在用比诱导95%生长抑制的浓度高10倍的药物浓度处理后才增加。在用星形孢菌素处理的培养物中,在15 - 92%生长抑制范围内,ELISA吸光度值与细胞毒性之间存在近似线性关系。用细胞毒性浓度的顺铂处理的乳腺癌细胞中缺乏凋亡信号,表明该药物通过非凋亡机制杀死细胞,而凋亡是星形孢菌素导致细胞死亡的主要机制。这里描述的甲酰胺 - MAb凋亡ELISA可能为基于药物诱导或抑制凋亡能力的高通量药物筛选提供基础。