Nie Yong-Zhan, He Feng-Tian, Li Zhi-Kui, Wu Kai-Chun, Cao Yun-Xin, Chen Bao-Jun, Fan Dai-Ming
Institute of Digestive Diseases,Xijing Hospital, Fourth Military Medical University, Changle West Road, Xi'an 710032,Shannxi Province China.
World J Gastroenterol. 2002 Aug;8(4):619-23. doi: 10.3748/wjg.v8.i4.619.
To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens.
The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E.coli HB2151 to express soluble ScFv.
The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60 %, 24 % and 30 %. MC3-ScFv had M(r) 32 000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab.
The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies.
研究以固定化全细胞作为单克隆抗体结合抗原的载体淘选和筛选抗肿瘤单克隆抗体噬菌体展示重组单链可变片段（ScFv）的可行性。
构建抗结直肠癌单克隆抗体MC3Ab、MC5Ab及抗胃癌单克隆抗体MGD1的重组噬菌体展示文库。通过对表达单克隆抗体结合抗原的结直肠癌和胃癌细胞进行改良固定来进行淘选和筛选。分析噬菌体克隆与亲本抗体对肿瘤细胞结合特异性的一致性。用竞争ELISA鉴定阳性克隆的噬菌体，并感染大肠杆菌HB2151以表达可溶性ScFv。
MC3-ScF-MC5-ScFv和MGD1-ScFv阳性克隆比例分别为60%、24%和30%。经Western印迹证实MC3-ScFv的相对分子质量为32 000。4种阳性重组噬菌体抗体与MC3Ab对抗原的特异性无差异。
改良的全肿瘤细胞固定方法用于淘选和筛选抗肿瘤单克隆抗体噬菌体展示ScFv高效、便捷且可行。
