Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout,ACS Applied Bio Materials

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Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout
ACS Applied Bio Materials ( IF 4.6 ) Pub Date : 2021-04-06 , DOI: 10.1021/acsabm.0c01680
Jaeyong Jung ₁ , Ji-Hong Bong ₁ , Soo Jeong Lee ₁ , Moon-Ju Kim ₁ , Jeong Soo Sung ₁ , Misu Lee ₂ , Min-Jung Kang ₃ , Jungsik Song ₄ , Joachim Jose ₅ , Jae-Chul Pyun ₁

Affiliation

To date, medical diagnosis of gout and pseudogout has been performed by observing the crystals in the joint fluid of patients under a polarized microscope. Conventional diagnostic methods using a polarized microscope have disadvantages, such as time-consuming analysis, a high false negative rate, and difficulty in distinguishing gout with monosodium urate (MSU) crystals and pseudogout with calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluids. In this study, a chromogenic assay for the diagnosis of gout and pseudogout, without the requirement of a polarized microscope and trained experts, was proposed using Fv antibodies with specific binding activities to MSU and CPPD crystals. The IgG V_H chain Fv library with randomized complementarity-determining region 3 (CDR3) region was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv antibodies with binding activity to MSU and CPPD crystals were screened from the autodisplayed Fv library on the E. coli outer membrane, and five clones were selected. On the basis of the binding properties of the screened Fv antibodies, peptides with the selected clone of amino acid sequences of the CDR3 region (15 residues) were chemically synthesized. The binding properties of the synthetic peptides with amino acid sequences of CDR3 regions from the selected clones were analyzed using fluorescence imaging and flow cytometry, and the affinity constants (K_d) of each peptide for binding to MSU and CPPD crystals were calculated by fitting based on the isotherm model. A chromogenic assay configuration for gout and pseudogout was developed using synthetic peptides. In this chromogenic assay, synthetic peptides labeled with biotin and streptavidin–horseradish peroxidase (HRP) complex were used, and crystal detection was possible using a chromogenic reaction between HRP and a chromogenic substrate (TMB). Finally, gout and pseudogout were diagnosed by detecting MSU and CPPD crystals in the synovial fluid in the concentration range of 0–300 μg/mL.

中文翻译：

筛选对尿酸单钠和焦磷酸钙二水合物晶体具有特异性结合活性的 Fv 抗体用于诊断痛风和假性痛风

迄今为止，已通过在偏光显微镜下观察患者关节液中的晶体来进行痛风和假痛风的医学诊断。使用偏光显微镜的常规诊断方法存在分析耗时、假阴性率高、难以区分痛风与尿酸单钠 (MSU) 晶体和滑液中焦磷酸钙 (CPPD) 晶体的假性痛风等缺点。在这项研究中，提出了一种用于诊断痛风和假痛风的显色测定法，无需偏光显微镜和训练有素的专家，使用对 MSU 和 CPPD 晶体具有特异性结合活性的 Fv 抗体。IgG V _H使用自动展示技术在大肠杆菌的外膜上表达具有随机互补决定区 3 (CDR3) 区的链 Fv 文库。从大肠杆菌外膜自动展示的Fv文库中筛选出与MSU和CPPD晶体具有结合活性的靶Fv抗体，筛选出5个克隆。基于筛选的 Fv 抗体的结合特性，化学合成具有 CDR3 区氨基酸序列（15 个残基）的选定克隆的肽。使用荧光成像和流式细胞术分析合成肽与来自所选克隆的 CDR3 区氨基酸序列的结合特性，以及亲和常数 ( K _d) 与 MSU 和 CPPD 晶体结合的每种肽的含量通过基于等温线模型的拟合来计算。使用合成肽开发了用于痛风和假痛风的显色测定配置。在该显色测定中，使用了用生物素和链霉亲和素-辣根过氧化物酶 (HRP) 复合物标记的合成肽，并且可以使用 HRP 和显色底物 (TMB) 之间的显色反应进行晶体检测。最后通过检测滑液中浓度范围为0-300 μg/mL的MSU和CPPD晶体诊断痛风和假痛风。

更新日期：2021-04-19

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