引导选择抗细胞表面分子单链抗体

Isolation of Single Chain Antibodies Against Cell Surface Molecules by Pathfinder Selection

目的 用引导选择方法从抗KG1a细胞的噬菌体展示抗体库中分离单链抗体。方法 首先用KG1a细胞吸附法富集抗体库3轮或未富集,再分别用抗CD34单克隆抗体为引导分子进行引导选择,分离单克隆后用免疫荧光和流式细胞术鉴别其细胞结合特异性。酶联免疫吸附法分析部分克隆与CD34抗原的结合,并对10个阳性克隆进行DNA序列分析。用一级结构不同的单链抗体作免疫印迹,鉴别其抗原分子量。结果 从144个经3轮富集和引导选择的单菌落中挑选出100个阳性单克隆,从96个未经富集的文库中引导选择出2个阳性克隆。102个阳性克隆中47个可识别KG1a、HL60、U937、和CEM细胞(:KG1a+、HL60+、U937+、CEM+),55个只识别KG1a细胞(KG1a+、HL60-、U937-、CEM-)。其中28个只识别KG1a细胞的单克隆经ELISA检测均不结合CD34抗原。10个阳性克隆的DNA序列分析显示来自4种不同克隆,所结合的抗原显示不同的分子量。结论从抗KG1a单链抗体库分出102个能识别造血干细胞和祖细胞的阳性克隆,引导选择在改进筛选效率方面具有优越性。

Objective To isolate single chain antibody fragments (scFv) against cell surface molecules by pathfinder selection from an anti-KG1a cell scFv phage library. Methods The anti-KG1a scFv library was enriched by KG1a cell panning for three rounds, or unenriched, then processed for pathfinder selection respectively using anti-CD34 monoclonal antibody as pathfinder molecule. ScFv phage clones were randomly picked and identified by binding KG1a cells using immunofluorescein and flow cytometry. The KG1a+ clones were further identified by KG1a, HL60, U937, and CEM cell lines and ELISA. Their antigenic molecules on cell surface were digested by chymopapain and analyzed by flow cytometry. DNAs from ten positive clones were sequenced. The scFv clones with different primary structure were used to analyze the molecular weight of their antigens by Western blot. Results One hundred and two KG1a+ scFv phage clones were isolated from 144 enriched and 96 unenriched scFv phage library respectively, among which 47 bound KG1a, HL60, U937, and CEM cells, 55 bound KGla cells exclusively. None of 28 KG1a+, HL60-, U937- , and CEM- scFv clones bound to the CD34 antigen, as confirmed by ELISA, although most of their antigens were sensitive to chymopapain digestion. DNA sequences from ten positive clones showed that they were from four different clones. They bound antigens with different molecular weight. Conclusions One hundred and two scFv phage clones specific for hematopoietic stem and progenitor cells have been isolated from an anti-KG1a cell scFv phage library. The pathfinder selection has showed advantages to improve the screening efficacy of scFv phage clones against antigens, which present at very low densities on the cell surface.