Li Hong-bin, Zhang Xuan, Tang Fu-lin, Zhang Feng-chun
Department of Rheumatology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100032, China.
Zhonghua Nei Ke Za Zhi. 2010 Jul;49(7):614-7.
To obtain the soluble single-chain fragment V (ScFv) monoclonal antibodies (McAbs) against the SSA antigen epitopes.
Three octapeptides (60000 SSA antigen residues 482-493 termed as P1 epitope, residues 310-323 termed as P2 epitope and residues 230-241 termed as P3 epitope) were synthesized on the lysine frame. The McAbs were panned by coating the corresponding as targets. The specificity, affinity and gene sequences of the positive clones were assessed. Soluble single-chain fragment V antibodies special for SSA antigen epitopes were expressed and then identified.
After 5 rounds of panning, reactive scFv clones contained full-length scFv antibodies coding regions were obtained, with sufficient affinity and specificity for respective antigen peptides. The absorbance values at 410 nm of the fusion protein of anti-P1-P3 activity with the corresponding peptides were 1.43±0.23, 0.82±0.31 and 0.80±0.25, and there was also statistically significant difference in the cross reactions (P<0.01). Three clones were successfully expressed and then purified by His-bind resin. The activity in vivo of soluble ScFv antibodies was identified to be positive by the indirect immune-fluorescence assay on Hep-2 cells.
Soluble ScFv McAbs against corresponding SSA antigen peptides with high affinity, specificity and activity in vivo were obtained, which are to be competent enough for epitopes expression on the target organs.
获取针对SSA抗原表位的可溶性单链抗体片段（ScFv）单克隆抗体（McAbs）。
在赖氨酸框架上合成三种八肽（60000 SSA抗原的482 - 493位残基称为P1表位，310 - 323位残基称为P2表位，230 - 241位残基称为P3表位）。以相应包被物为靶点进行单克隆抗体淘选。评估阳性克隆的特异性、亲和力和基因序列。表达并鉴定针对SSA抗原表位的可溶性单链抗体片段。
经过5轮淘选，获得了包含全长单链抗体片段编码区的反应性单链抗体片段克隆，对各自的抗原肽具有足够的亲和力和特异性。抗P1 - P3活性的融合蛋白与相应肽段在410 nm处的吸光度值分别为1.43±0.23、0.82±0.31和0.80±0.25，交叉反应也有统计学显著差异（P<0.01）。成功表达了三个克隆并通过His - bind树脂进行纯化。通过对Hep - 2细胞的间接免疫荧光测定，鉴定可溶性单链抗体片段在体内的活性为阳性。
获得了针对相应SSA抗原肽的具有高亲和力、特异性和体内活性的可溶性单链抗体片段单克隆抗体，足以在靶器官上表达表位。
