RESUMO
The conserved coronavirus fusion peptide is a target of broadly neutralizing antibodies.
Assuntos
Alphacoronavirus , Anticorpos Antivirais , Betacoronavirus , Anticorpos Amplamente Neutralizantes , Peptídeos , Glicoproteína da Espícula de Coronavírus , Proteínas Virais de Fusão , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/imunologia , Peptídeos/química , Peptídeos/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Testes de Neutralização , Humanos , Betacoronavirus/imunologia , Alphacoronavirus/imunologia , Complexo Antígeno-Anticorpo/química , Infecções por Coronavirus/prevenção & controleRESUMO
As of July 22, 2020, more than 14.7 million infections of SARS-CoV-2, the virus responsible for Coronavirus Disease 2019 (COVID-19), have been confirmed globally. Serological assays are essential for community screening, assessing infection prevalence, aiding identification of infected patients, and enacting appropriate treatment and quarantine protocols in the battle against this rapidly expanding pandemic. Antibody detection by agglutination-PCR (ADAP) is a pure solution phase immunoassay that generates a PCR amplifiable signal when patient antibodies agglutinate DNA-barcoded antigen probes into a dense immune complex. Here, we present an ultrasensitive and high-throughput automated liquid biopsy assay based on the Hamilton Microlab ADAP STAR automated liquid-handling platform, which was developed and validated for the qualitative detection of total antibodies against spike protein 1 (S1) of SARS-CoV-2 that uses as little as 4 µL of serum. To assess the clinical performance of the ADAP assay, 57 PCR-confirmed COVID-19 patients and 223 control patients were tested. The assay showed a sensitivity of 98% (56/57) and a specificity of 99.55% (222/223). Notably, the SARS-CoV-2-negative control patients included individuals with other common coronaviral infections, such as CoV-NL63 and CoV-HKU, which did not cross-react. In addition to high performance, the hands-free automated workstation enabled high-throughput sample processing to reduce screening workload while helping to minimize analyst contact with biohazardous samples. Therefore, the ADAP STAR liquid-handling workstation can be used as a valuable tool to address the COVID-19 global pandemic.
Assuntos
Alphacoronavirus/imunologia , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Coronavirus Humano NL63/imunologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Automação Laboratorial , Quirópteros , Técnicas de Laboratório Clínico , Reações Cruzadas , Ensaios de Triagem em Larga Escala , Humanos , Imunoensaio , Pandemias , Reação em Cadeia da Polimerase , Procedimentos Cirúrgicos Robóticos , Sensibilidade e EspecificidadeRESUMO
A swine acute diarrhea syndrome coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in Southern China in 2017. To develop an antigen that is specific, sensitive, and easy to prepare for serological diagnosis, antigenic sites in the SADS-CoV nucleocapsid (N) protein were screened. We generated and characterized an N-reactive monoclonal antibody (mAb) 3E9 from mice immunized with recombinant N protein. Through fine epitope mapping of mAb 3E9 using a panel of eukaryotic-expressed polypeptides with GFP-tags, we identified the motif 343DAPVFTPAP351 as the minimal unit of the linear B-cell epitope recognized by mAb 3E9. Protein sequence alignment indicated that 343DAPVFTPAP351 was highly conserved in different SADS-CoV strains and SADS-related coronaviruses from bat, with one substitution in this motif in HKU2-related bat coronavirus. Using mAb 3E9, we observed that N protein was expressed in the cytoplasm and was in the nucleolus during SADS-CoV replication. N protein was immunoprecipitated from SADS-CoV-infected Vero E6 cells. Taken together, our results indicated that 3E9 mAb could be a useful tool to investigate the structure and function of N protein during viral replication.