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      Preparation of highly specific monoclonal antibodies against SARS‐CoV‐2 nucleocapsid protein and the preliminary development of antigen detection test strips

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          Abstract

          The coronavirus disease 2019 (COVID‐19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS‐CoV‐2, constructing the pET‐28a (+)‐N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS‐CoV‐2‐N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high‐titer and high‐specificity monoclonal antibodies, and the test strips exclusively react with the SARS‐CoV‐2‐N protein and no cross‐reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS‐CoV‐2 infection through serological antigen.

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          Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus–Infected Pneumonia in Wuhan, China

          In December 2019, novel coronavirus (2019-nCoV)-infected pneumonia (NCIP) occurred in Wuhan, China. The number of cases has increased rapidly but information on the clinical characteristics of affected patients is limited.
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            Virological assessment of hospitalized patients with COVID-2019

            Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
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              Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS‐CoV‐2 infection diagnosis

              Abstract The outbreak of the novel coronavirus disease (COVID‐19) quickly spread all over China and to more than 20 other countries. Although the virus (severe acute respiratory syndrome coronavirus [SARS‐Cov‐2]) nucleic acid real‐time polymerase chain reaction (PCR) test has become the standard method for diagnosis of SARS‐CoV‐2 infection, these real‐time PCR test kits have many limitations. In addition, high false‐negative rates were reported. There is an urgent need for an accurate and rapid test method to quickly identify a large number of infected patients and asymptomatic carriers to prevent virus transmission and assure timely treatment of patients. We have developed a rapid and simple point‐of‐care lateral flow immunoassay that can detect immunoglobulin M (IgM) and IgG antibodies simultaneously against SARS‐CoV‐2 virus in human blood within 15 minutes which can detect patients at different infection stages. With this test kit, we carried out clinical studies to validate its clinical efficacy uses. The clinical detection sensitivity and specificity of this test were measured using blood samples collected from 397 PCR confirmed COVID‐19 patients and 128 negative patients at eight different clinical sites. The overall testing sensitivity was 88.66% and specificity was 90.63%. In addition, we evaluated clinical diagnosis results obtained from different types of venous and fingerstick blood samples. The results indicated great detection consistency among samples from fingerstick blood, serum and plasma of venous blood. The IgM‐IgG combined assay has better utility and sensitivity compared with a single IgM or IgG test. It can be used for the rapid screening of SARS‐CoV‐2 carriers, symptomatic or asymptomatic, in hospitals, clinics, and test laboratories.
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                Author and article information

                Contributors
                hjlv61@126.com
                Journal
                J Med Virol
                J Med Virol
                10.1002/(ISSN)1096-9071
                JMV
                Journal of Medical Virology
                John Wiley and Sons Inc. (Hoboken )
                0146-6615
                1096-9071
                21 December 2021
                April 2022
                : 94
                : 4 , Special Issue on New Coronavirus (2019‐nCoV or SARS‐CoV‐2) and the Outbreak of the Respiratory Illness (COVID‐19): Part‐XIX ( doiID: 10.1002/jmv.v94.4 )
                : 1633-1640
                Affiliations
                [ 1 ] Institute of Parasitic Disease Hangzhou Medical College Hangzhou China
                [ 2 ] The Key Laboratory of Blood Safety Research, Blood Center of Zhejiang Province Hangzhou China
                Author notes
                [*] [* ] Correspondence Hangjun Lv, Institute of Parasitic Disease, Hangzhou Medical College, Hangzhou 310013, China.

                Email: hjlv61@ 123456126.com

                Author information
                http://orcid.org/0000-0002-4596-7026
                Article
                JMV27520
                10.1002/jmv.27520
                9303534
                34904253
                794ab1e0-8fd5-410f-ad01-379f1f9df2bd
                © 2021 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 22 November 2021
                : 07 October 2021
                : 10 December 2021
                Page count
                Figures: 8, Tables: 0, Pages: 8, Words: 4314
                Funding
                Funded by: Provincial Key R & D program of Zhejiang Department of Science and Technology
                Award ID: 2019C03057
                Funded by: Zhejiang Provincial fund–Qingshanhu science and Technology City Joint Fund
                Award ID: LQY19H190002
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                April 2022
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.1.7 mode:remove_FC converted:21.07.2022

                Microbiology & Virology
                colloidal gold immunochromatography,delta (b.1.617.2) variant,recombinant sars‐cov‐2 nucleocapsid protein,sars‐cov‐2,the monoclonal antibodies

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