Shengli Clinical Medical College of Fujian Medical University, Fujian Provincial Hospital, Fuzhou, China.
Department of Pharmacy, Fujian Provincial Hospital, Fuzhou, China.
Front Immunol. 2023 Jun 16;14:1127849. doi: 10.3389/fimmu.2023.1127849. eCollection 2023.
Scleroderma 70 (Scl-70) is commonly used in the clinic for aiding systemic sclerosis (SSc) diagnosis due to its recognition as autoantibodies in the serum of SSc patients. However, obtaining sera positive for anti-Scl-70 antibody can be challenging; therefore, there is an urgent need to develop a specific, sensitive, and easily available reference for SSc diagnosis. In this study, murine-sourced scFv library were screened by phage display technology against human Scl-70, and the scFvs with high affinity were constructed into humanized antibodies for clinical application. Finally, ten high-affinity scFv fragments were obtained. Three fragments (2A, 2AB, and 2HD) were select for humanization. The physicochemical properties of the amino acid sequence, three-dimensional structural basis, and electrostatic potential distribution of the protein surface of different scFv fragments revealed differences in the electrostatic potential of their CDR regions determined their affinity for Scl-70 and expression. Notably, the specificity test showed the half-maximal effective concentration values of the three humanized antibodies were lower than that of positive patient serum. Moreover, these humanized antibodies showed high specificity for Scl-70 in diagnostic immunoassays for ANA. Among these three antibodies, 2A exhibited most positive electrostatic potential on the surface of the CDRs and highest affinity and specificity for Scl-70 but with least expression level; thus, it may provide new foundations for developing enhanced diagnostic strategies for SSc.
硬皮病 70（Scl-70）通常在临床上用于辅助系统性硬皮病（SSc）的诊断，因为它被认为是 SSc 患者血清中的自身抗体。然而，获得抗 Scl-70 抗体阳性的血清可能具有挑战性；因此，迫切需要开发一种特异性、敏感性和易于获得的 SSc 诊断参考物。在这项研究中，通过噬菌体展示技术筛选了针对人 Scl-70 的鼠源 scFv 文库，并将高亲和力的 scFv 构建成可用于临床应用的人源化抗体。最终获得了十个高亲和力的 scFv 片段。选择三个片段（2A、2AB 和 2HD）进行人源化。不同 scFv 片段的氨基酸序列理化性质、三维结构基础和蛋白质表面的静电势分布揭示了其 CDR 区域静电势的差异，决定了它们与 Scl-70 的亲和力和表达。值得注意的是，特异性测试表明，三种人源化抗体的半数有效浓度值均低于阳性患者血清。此外，这些人源化抗体在 ANA 的诊断免疫测定中对 Scl-70 具有高度特异性。在这三种抗体中，2A 在 CDR 表面表现出最强的正静电势和对 Scl-70 的最高亲和力和特异性，但表达水平最低；因此，它可能为开发增强的 SSc 诊断策略提供新的基础。
