Department of Dermatology, Xijing Hospital, Fourth Military Medical University, 127 Changlexi Road, Xi'an 710032, China.
J Dermatol Sci. 2013 Oct;72(1):25-31. doi: 10.1016/j.jdermsci.2013.05.010. Epub 2013 Jun 13.
Pathogenic autoantibodies in bullous pemphigoid (BP) recognize the non-collagenous 16A domain (NC16A) of collagen XVII (COL17), a hemidesmosomal component at the skin membrane. This immune inflammation involves activation of the complement cascade via the classical pathway. With similar antigen binding activity, Fab and single-chain variable fragments (scFv) of pathogenic anti-COL17 antibodies can interfere with COL17 binding of autoantibodies, blocking subsequent complement activation and granulocyte activation.
To characterize the biological functions of human anti-COL17 scFv antibody.
We constructed scFv antibodies against the corresponding antigen from parental Fab by expression in Escherichia coli. IgG autoantibodies against COL17 were purified by affinity chromatography from serum of BP patients. The inhibitory effects of anti-COL17 scFv on binding of BP autoantibodies to the NC16A domain of human COL17 antigen were observed by inhibition ELISA, immunofluorescence, and inhibition of complement activation. Reactive oxygen production assay and BP cryosection model were performed to assess the inhibitory effect of scFv on granulocyte activation and then the dermal-epidermal separation.
ELISA and Western blot showed specific binding of scFv to COL17. We found that anti-COL17 scFv can inhibit the binding of intact IgG purified from BP parents to the corresponding COL17 antigen and then subsequent C1q and C3 activation and granulocyte activation in vitro. Most importantly, we confirmed that recombinant scFv can inhibit BP-IgG induced dermal-epidermal separation by BP cryosection model.
The anti-COL17 scFv antibody can inhibit the binding of BP-IgG autoantibodies to COL17, thereby affecting subsequent complement activation and granulocyte activation in vitro. Our results suggest that blocking pathogenic epitopes using engineered scFv is an efficient BP therapy.
大疱性类天疱疮（BP）中的致病自身抗体识别半桥粒膜成分 XVII 型胶原（COL17）的非胶原 16A 结构域（NC16A）。这种免疫炎症涉及经典途径补体级联的激活。具有相似抗原结合活性的致病抗 COL17 抗体的 Fab 和单链可变片段（scFv）可干扰自身抗体与 COL17 的结合，从而阻断随后的补体激活和粒细胞激活。
研究人抗 COL17 scFv 抗体的生物学功能。
我们通过在大肠杆菌中表达，从亲本 Fab 构建针对相应抗原的 scFv 抗体。通过亲和层析从 BP 患者的血清中纯化针对 COL17 的 IgG 自身抗体。通过抑制 ELISA、免疫荧光和抑制补体激活来观察抗 COL17 scFv 对 BP 自身抗体与 COL17 抗原的 NC16A 结构域结合的抑制作用。进行活性氧产生测定和 BP 冷冻切片模型，以评估 scFv 对粒细胞激活和随后表皮-真皮分离的抑制作用。
ELISA 和 Western blot 显示 scFv 特异性结合 COL17。我们发现抗 COL17 scFv 可抑制从 BP 亲本中纯化的完整 IgG 与相应 COL17 抗原的结合，然后抑制体外的 C1q 和 C3 激活以及粒细胞激活。最重要的是，我们通过 BP 冷冻切片模型证实重组 scFv 可抑制 BP-IgG 诱导的表皮-真皮分离。
抗 COL17 scFv 抗体可抑制 BP-IgG 自身抗体与 COL17 的结合，从而影响体外补体的后续激活和粒细胞激活。我们的结果表明，使用工程 scFv 阻断致病表位是一种有效的 BP 治疗方法。
