Anti-coronavirusdisease-2019 (COVID-19; anti-severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2)) antibodies against nucleoprotein (NP) were purified from pig sera. Following the separation of the antibody fraction using a protein-A column, the final yield of the purified antibodies against SARS-CoV-2 NPs was estimated to be 0.26 ± 0.05 % (absolute amount of 143.4 ± 25.2 ng, n=5) from 1 mL of pig sera. The binding activities of the isolated antibodies were confirmed using immunoassay and immunostaining. Based on the specific binding activity to NPs, a quantitative assay was performed using a surface plasmon resonance (SPR) biosensor. From the doseresponse curve, the binding constant (Kd) was calculated to be 185 pM and the limit of detection was estimated to be 1.02 pM. The SPR biosensor with the isolated antibodies against SARS-CoV-2 NPs was applied for the detection of SARS-CoV-2, MERS-CoV, and CoV strain 229E in culture fluid.

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 (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.

Anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) nucleoprotein (NP) antibodies were isolated from pig sera using human SARS-CoV-2 NP-immobilized magnetic beads. The binding properties of the isolated antibodies against SARS-CoV-2 NP were tested via flow cytometry using SARS-CoV-2 NP-immobilized magnetic beads. A competitive immunoassay was developed for detecting SARS-CoV-2 NP as well as SARS-CoV-2 in the culture fluid using magnetic beads with immobilized anti-SARS-CoV-2 NP antibodies. Selectivity tests were carried out during the competitive immunoassay for SARS-CoV, MERS-CoV, and CoV strain 229E in the culture fluid.

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