Wang M, Nilsson G, Carlberg M, Dricu A, Wejde J, Kreicbergs A, Larsson O
Department of Tumour Pathology, Karolinska Institutet, Stockholm, Sweden.
Virchows Arch. 1998 Feb;432(2):131-4. doi: 10.1007/s004280050146.
We applied Western blotting, using an antibody against the carboxy terminal of the FLI1 protein, for detection of the 68-kDa EWS/FLI1 fusion protein in cultured Ewing's sarcoma cells and in four surgical biopsies of Ewing's sarcoma. Of six different human cell lines, the 68-kDa fusion protein was identified only in Ewing's sarcoma cells carrying the t(11;22)(q24;q12) translocation. The four samples from Ewing's sarcoma patients were also found to contain the 68-kDa fusion protein. The lowest detection level for total protein loaded on the gel was 0.3 microg. When whole Ewing's sarcoma cells were used for Western blotting without prior protein extraction, the lowest detection level was 1,300 cells. It will be possible to use Western blotting for detection of the EWS/FLI1 fusion protein in the diagnosis of Ewing's sarcoma in surgical biopsy specimens, and possibly also in fine-needle aspirates. As the method is not dependent on the quality of mRNA in the sample and involves no risk of contamination, it will be a powerful complement to the reverse-transcription polymerase chain reaction (RT-PCR).
我们运用蛋白质免疫印迹法，使用针对FLI1蛋白羧基末端的抗体，来检测培养的尤因肉瘤细胞以及尤因肉瘤的四份手术活检样本中68 kDa的EWS/FLI1融合蛋白。在六种不同的人类细胞系中，仅在携带t(11;22)(q24;q12)易位的尤因肉瘤细胞中鉴定出了68 kDa的融合蛋白。尤因肉瘤患者的四份样本也被发现含有68 kDa的融合蛋白。加载到凝胶上的总蛋白的最低检测水平为0.3微克。当未经预先蛋白质提取就使用完整的尤因肉瘤细胞进行蛋白质免疫印迹法时，最低检测水平为1300个细胞。蛋白质免疫印迹法可用于检测手术活检样本中尤因肉瘤诊断时的EWS/FLI1融合蛋白，也可能用于细针穿刺抽吸样本。由于该方法不依赖于样本中mRNA的质量且不存在污染风险，它将成为逆转录聚合酶链反应（RT-PCR）的有力补充。
