Construction and characterization of a single-chain antibody fragment derived from thymus of a patient with myasthenia gravis
Construction and characterization of a single-chain antibody fragment derived from thymus of a patient with myasthenia gravis
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重症肌无力患者胸腺来源的单链抗体片段的构建和表征
DOI：
10.1080/08916930290016646
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发表时间：
2002-03-01
期刊：
影响因子：
3.5
通讯作者：
De Baets, MH
中科院分区：
文献类型：
--
作者：
Meng, FP;Stassen, MHW;De Baets, MH
重症肌无力（MG）和相应的实验性自身免疫性重症肌无力（EAMG）动物模型中的致病性抗乙酰胆碱受体（AChR）抗体主要识别AChR的主要免疫原性区域（MIR）。二价抗MIR抗体与AChR的α亚基结合，通过抗原调节和补体激活导致AChR损失。致病性抗AChR抗体的单价Fab和单链可变片段（scFv）可干扰致病性抗体的AChR结合。在本研究中，scFv637构建从其亲本Fab637，先前分离的胸腺衍生的噬菌体展示库与特异性对人AChR（hAChR）的抗MIR，通过PCR扩增。在标准沉淀放射免疫分析（RIA）中，细菌产生的scFv637能够与hAChR结合。免疫组织化学染色显示，ScFv637还与猴神经肌肉接头上的猴AChR原位结合。此外，scFv637能够抑制其完整IgG637和抗MIR mAb 35与hAChR的结合，在竞争性ELISA中分别高达32.9%和73.0%，并且在竞争性RIA中抑制MG患者血清高达45.5%。因此，与亲本Fab 637相比，scFv637更容易操作，亲和力和稳定性更高，可作为MG特异性免疫治疗的候选药物。
Pathogenic anti-acetylcholine receptor (AChR) antibodies in myasthenia gravis (MG) and the corresponding animal model, experimental autoimmune myasthenia gravis (EAMG), principally recognize the main immunogenic region (MIR) of the AChR. Bivalent anti-MIR antibodies binding to the alpha-subunits of AChR result in AChR loss by antigenic modulation and complement activation. Monovalent Fab and single-chain variable fragments (scFv) of pathogenic anti-AChR antibodies can interfere with AChR binding of the pathogenic antibodies. In the present study, scFv637 was constructed from its parental Fab637, previously isolated from a thymus-derived phage display library with specificity toward anti-MIR of human AChR (hAChR), by PCR amplification. Bacterial produced scFv637 was able to bind to hAChR in standard precipitation radioimmunoassay (RIA). ScFv637 also bound to monkey AChR in situ on monkey neuromuscular junctions as showed in immunohistochemical staining. Furthermore, scFv637 was capable of inhibiting the binding of its intact IgG637 and anti-MIR mAb35 binding to hAChR up to 32.9 and 73.0%, respectively demonstrated in a competitive ELISA, and of MG patient sera from up to 45.5% in a competitive RIA. Therefore, scFv637, easier for manipulation in improvement of affinity and stability compared with its parental Fab637, may serve as an alternative candidate for specific immunotherapy in MG.