研究目的
To develop a sensitive approach for detecting leukemic cancer stem cells (LSCs) based on a conjugate of an anti-CD45 scFv and quantum dot.
研究成果
The study successfully developed a low-cost and efficient approach for the detection of CD45RA positive cells, including LSCs, using an anti-CD45RA scFv conjugated with C dots. This method showed high specificity and binding ability to CD45RA expressing cells, offering a promising tool for monitoring therapy response in AML patients.
研究不足
The study focused on the detection of CD45RA positive cells using a specific scFv and C dots conjugate. The biological activity of the scFv in AML patients compared to healthy donors was not assessed, indicating a need for future research in this area.
1：Experimental Design and Method Selection:
The study involved the construction of an anti-CD45RA scFv from hybridoma cells, its expression in Escherichia coli, purification, and conjugation with carbon dots (C dots) for the detection of CD45RA positive cells.
2：Sample Selection and Data Sources:
Jurkat cell line as CD45RA+ cells and K562 cell line as CD45RA- cells were used.
3：List of Experimental Equipment and Materials:
Anti-CD45RA hybridoma cell, Escherichia coli NovaBlue GigaSingles?, Escherichia coli Rosetta (DE3) cells, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-morpholinoethansulfonic acid monohydrate (MES), Qiaquick Gel Extraction Kit, pfu High Fidelity DNA polymerase, pET32Ek/LIC vector, FACS Calibur flow cytometer.
4：Experimental Procedures and Operational Workflow:
The scFv was constructed, expressed, purified, and conjugated with C dots. The conjugate was then used for the detection of CD45RA positive cells via flow cytometry and ICC.
5：Data Analysis Methods:
The binding specificity of the scFv to CD45RA was analyzed by flow cytometry and fluorescence microscopy.
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