Jung, J. Bong, J.-H. Lee, S.J. Kim, M.-J. Sung, J.S. Lee, M. Kang, M.-J. Song, J. Jose, J. Pyun, J.-C. 2024-01-19T15:03:39Z 2024-01-19T15:03:39Z 2021-09-02 2021-04 2576-6422 https://pubs.kist.re.kr/handle/201004/117235 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 VH 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 (Kd) 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. ？ 2021 American Chemical Society. All rights reserved. English American Chemical Society Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout Article 10.1021/acsabm.0c01680 1 ACS Applied Bio Materials, v.4, no.4, pp.3388 - 3397 ACS Applied Bio Materials 4 4 3388 3397 N scopus 2-s2.0-85105107555 Article Amino acids Antibodies Binding energy Calcium compounds Chromogenics Cloning Diagnosis Escherichia coli Fluorescence imaging Microscopes Peptides Calcium pyrophosphate Chromogenic substrate Complementarity-determining region 3 Concentration ranges Horseradish peroxidase Polarized microscope Specific binding activity Time consuming analysis Crystals antibody library autodisplay calcium pyrophosphate dehydrate diagnosis gout monosodium urate pseudogout