Enzyme-linked immunosorbent assay (ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening.
Enzyme-linked immunosorbent assay (ELISA) for the specific detection of apoptotic cells and its application to rapid drug screening.
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DOI：
10.1016/s0022-1759(01)00387-8
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发表时间：
2001-07-01
影响因子：
2.2
通讯作者：
Krishan, A
中科院分区：
文献类型：
--
作者：
Frankfurt, OS;Krishan, A
我们建立了一种用于检测凋亡细胞的特异性和敏感性的固相酶联免疫吸附试验。这种方法是基于抗单链DNA(SsDNA)的单抗(MAb)特异性识别凋亡细胞的能力。该方法包括将细胞与96孔板结合，用甲酰胺处理附着的细胞以变性凋亡细胞中的DNA，并用抗SSDNA单抗和过氧化物酶标记的抗鼠IgM的混合物一步法对变性DNA进行染色。当凋亡细胞的数量从500个增加到5000个时，信号几乎呈线性增加。未经处理和坏死的细胞或由过氧化氢诱导的单链DNA断裂的细胞不产生高于本底水平的信号。在培养的白血病细胞中，用ID50浓度的依托泊苷处理，在同一96孔板上染色和分析，检测到强烈的ELISA信号。依托泊苷处理的耐药细胞株和敏感细胞株的吸光度比值与生长抑制法测定的耐药程度一致。这些数据表明，该酶联免疫吸附试验具有足够的敏感性，可用于药物筛选。在顺铂处理的乳腺癌细胞培养中，只有在药物浓度高于导致95%生长抑制的浓度10倍后，ELISA值才会增加。在用星形孢子素处理的培养物中。在细胞生长抑制率为15-92%的范围内，酶标法的吸光度与细胞毒性呈良好的线性关系。细胞毒浓度顺铂处理的乳腺癌细胞缺乏凋亡信号，表明该药物通过非凋亡机制杀死细胞，而细胞凋亡是星形孢菌素引起的细胞死亡的主要机制。本研究建立的甲酰胺-单抗细胞凋亡酶联免疫吸附试验可为高通量筛选具有诱导或抑制细胞凋亡能力的药物提供依据。(C)2001 Elsevier Science B.V.保留所有权利。
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 H2O2 did not produce signal above the background level. In leukemic cell cultures grown, treated with ID50 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. (C) 2001 Elsevier Science B.V. All rights reserved.