Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer
Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer
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DOI：
10.1093/protein/10.4.423
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发表时间：
1997-04-01
期刊：
影响因子：
--
通讯作者：
Hudson, PJ
中科院分区：
文献类型：
--
作者：
Kortt, AA;Lah, M;Hudson, PJ
利用10-残基（Gly(4)Ser）(2)和5 -残基（Gly(4)Ser）连接体连接抗神经氨酸酶抗体NC10的V-H和V-L结构域，构建了单链可变片段（scFvs）；将V-H结构域的c端残基连接到V-L结构域的n端，构建了零残基连接子scFv。具有10和5残基连接体的scFv仅形成二聚体抗体片段（M-r 52000），这些片段被证明是二价的，并且能够交联两个神经氨酸酶四聚体形成“三明治”型复合物；每个抗原结合位点也可以结合一个抗独特型Fab'。零残基连接体scFv （M-r 70 000）与三个活性抗原结合位点形成三聚体，每个位点结合一个抗独特型Fab'，产生一个M-r 212 000的复合物。然而，零残基连接体scFv的结合位点的取向使得它不能交联神经氨酸酶的四聚体。BIAcore生物传感器实验表明，当scFv固定在传感器表面时，10-和5 -残基连接体scFv二聚体和零残基连接体scFv三聚体中每个抗原结合位点的亲和力基本相同，但当scFv作为分析物时，由于多价scFv的结合性，二聚体和三聚体的结合亲和力明显增加。
Single-chain variable fragments (scFvs) of anti-neuraminidase antibody NC10 were constructed by joining the V-H and V-L domains with 10-residue (Gly(4)Ser)(2) and five-residue (Gly(4)Ser) linkers; a zero-residue linker scFv was constructed by joining the C-terminal residue of the V-H domain to the N-terminus of the V-L domain. The scFv with the 10- and five-residue linkers exclusively formed dimeric antibody fragments (M-r 52 000), These were shown to be bivalent and were able to cross-link two neuraminidase tetramers to form a 'sandwich' type complex; each antigen combining site could also bind an anti-idiotype Fab'. The zero-residue linker scFv (M-r 70 000) was shown to form a trimer with three active antigen combining sites, each binding an anti-idiotype Fab' to yield a complex of M-r 212 000. The orientation of the combining sites in the zero-residue linker scFv, however, was such that it could not cross-link tetramers of neuraminidase. BIAcore biosensor experiments showed that the affinity of each individual antigen combining site in both the 10- and five-residue linker scFv dimers and zero-residue linker scFv trimer was essentially the same when the scFvs were immobilized onto the sensor surface, However, when the scFvs were used as the analyte, the dimeric and trimeric scFvs showed an apparent increase in binding affinity due to the avidity of binding the multivalent scFvs.