Abstract

Leukemic cancer stem cells (LSCs), aberrantly overexpressing CD45RA are among the major causes of relapse following chemotherapy in patients with acute myeloid leukemia and serve as a highly sensitive marker for predicting relapse occurrence following chemotherapy. The main purpose of current study was to develop a sensitive approach for detecting LSCs based on a conjugate of an anti-CD45 scFv and quantum dot. The variable light and heavy chain sequences of a recently developed anti-CD45RA monoclonal antibody were derived from hybridoma cells and PCR amplified to construct scFv. Following insertion of scFv gene into a pET32a-lic vector and expression in Escherichia coli and purification, the purified scFv, was conjugated with carbon dots (C dots) and used for the detection of CD45RA ⁺cells while CD45RA-cells served as negative control. Subsequently, Functional activity of the conjugate was analyzed by flow cytometry and ICC to detect the cell surface antigen binding and detection ability. Based on results, purified CD45RA scFv conjugated C dots could specifically recognize CD45RA positive cells, but not any CD45RA negative ones. In conclusion, here we developed a low-cost but very efficient approach for detection of CD45RA positive cells including LSCs.

中文翻译：

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抗CD45RA-量子点缀合scFv检测白血病癌症干细胞的开发

摘要

异常表达CD45RA的白血病癌干细胞（LSC）是急性髓细胞性白血病患者化疗后复发的主要原因之一，并且是预测化疗后复发发生的高度敏感的标志物。当前研究的主要目的是开发一种基于抗CD45 scFv和量子点结合物的检测LSC的灵敏方法。最近开发的抗CD45RA单克隆抗体的可变轻链和重链序列来自杂交瘤细胞，并进行PCR扩增以构建scFv。将scFv基因插入pET32a-lic载体并在大肠杆菌中表达和纯化后，将纯化的scFv与碳点（C点）缀合，用于检测CD45RA ⁺CD45RA细胞用作阴性对照。随后，通过流式细胞术和ICC分析缀合物的功能活性，以检测细胞表面抗原结合和检测能力。根据结果​​，纯化的CD45RA scFv共轭C点可以特异性识别CD45RA阳性细胞，但不能识别任何CD45RA阴性细胞。总之，这里我们开发了一种低成本但非常有效的方法来检测CD45RA阳性细胞（包括LSC）。