Antibody Characteristics And Detection Methods In Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection

On January 30,2020,the World Health Organization announced the 2019 Corona Virus Disease 2019,(COVID-19)became the sixth international concern after HIN1 influenza A(2009),polio(2014),Ebola virus in West Africa(2014),Zika virus(2016)and Ebola virus in the Democratic Republic of the Congo(2019)of public health emergencies severely threaten global public health security.As of June 1,2020,the virus caused 6,057,853 people infection,resulting in 371,166 deaths.SARS-CoV-2 is an enveloped single-stranded positive-strand RNA virus belonging to the genus β-coronavirus.At present,the diagnosis of COVID-19 mainly uses PCR-based methods or deep sequencing to detect viral genomes,but these detection methods rely heavily on the presence of a sufficient number of viral genomes at the sampling site.Missing the window period of virus replication may cause false negatives,making infected patients have the possibility of spreading the virus.Increasing the detection rate is the key to improve the ability of epidemic prevention and control.It is especially important to develop a detection method that is faster,more convenient,and complementary to nucleic acid detection.Serological detection method is an important tool for pathogen detection,so it is a choice to construct detection methods for detecting SARS-CoV-2 antibody or antigen.The extent and time kinetics of the humoral response to SARS-CoV-2 are currently unclear.Although IgM antibody detection has been used in the diagnosis of COVID-19,the application value of IgM antibody detection,combined with PCR,are the detection sensitivity and accuracy improvement still needs further evaluate.Antigens are specific markers of viruses,which appear before antibodies in infected patients,detection of antigens can achieve the purpose of early diagnosis,but there was lack of research on SARS-CoV-2 antigen detection methods.Accordingly,we had established an antibody detection system based on the indirect enzyme-linked immunosorbent assay(ELISA)method.This method has the advantages of short experimental period,simple operation and low cost.According to the type of coating protein,both antibodies and antigens can be detected.In establishd the antigen detection system,we have adopted the double antibody sandwich ELIS A method,which had been applied to the detection of various viral antigens.First,the prokaryotic expression system was used to express the SARSCoV-2 nucleocapsid(N)protein.And the proteins were purified.The N protein was used to construct an ELISA detection method.The antibody composition and characteristics of suspected and confirmed cases from Wuhan,Beijing and other places were analyzed.A total of 208 plasma samples were collected from 82 confirmed cases and 58 suspected cases.The results showed that the median time for IgM and IgA antibody detection was 5 days(IQR 3-6),and IgG was 14 days after the onset of symptoms(IQR 10-18).the positive rates were 85.4%,92.7%,and 77.9%,respectively.In confirmed and suspected cases,the positive rates of IgM antibodies were 75.6%and 93.1%,respectively.5.5 days after the onset of symptoms,the detection efficiency of IgM ELISA was higher than that of qPCR.When the IgM ELISA test was used in combination with the qPCR method,the positive detection rate of each patient increased significantly(98.6%),And the positive detection rate using the qPCR method alone was 51.9%.Based on the above humoral immune response of SARS-CoV-2 can be used for the diagnosis of COVID-19.and it can significantly improve the detection rate when combined with the qPCR method.Subsequently,the method of double antibody sandwich ELISA for the detection of SARS-CoV-2 N protein was studied.The key point of this method is specific monoclonal antibodies against SARS-CoV-2.The amino acid similarity between SARS-CoV-2 and SARS-CoV N proteins was found to be 90.5%in the early stage,and they had highly similar antigenicity,so we speculate that anti-SARS-CoVN monoclonal antibody can react with SARS-CoV-2 N protein.Based on the previous research foundation of the research group,resuscitating the hybridoma cells constructed based on SARS-CoV N protein,two monoclonal antibodies A1 and A2 that can react with SARS-CoV-2 N protein were selected.A1 was more reactive with SARS-CoV-2 N protein.A double antibody sandwich ELISA method was established using anti-SARS-CoV-2 N protein polyclonal antibody,A1 and HRP-labeled anti-mouse IgG antibody,SARS-CoV-2 N protein and SARS-CoV-2 virus culture was used as a simulated antigen to determine the sensitivity,and the SARS-CoV-2 N protein were still detectable under 0.1 ng/μL without inactivation.The minimum detection amount of the virus culture was 20PFU.Double antibody sandwich ELISA was used to detect other coronavirus N protein.virus culture and pharyngeal swabs.This method has high specificity.The throat swabs,anal swabs and saliva samples collected from SARS-CoV-2 infected patients at different onset times were tested for antigens,and it was found that this method is suitable for the detection of throat swab samples.The sensitivity of the double antibody sandwich ELISA method still needs to be further optimized to increase the positive detection rate.In this study,we demonstrated the time kinetics of antibody response to SARS-CoV-2 in infected patients.We further demonstrated that combining the antibody testing with qPCR can significantly improve the diagnosis of COVID-19.A double antibody sandwich ELISA method for detecting SARS-CoV-2 N protein was constructed,which has potential application value.