| Background: Since December 2019,COVID-19 caused by SARS-CoV-2 has worldwidely spread,causing millions of deaths and becoming a serious threat to human lives.Accurate diagnosis of patients infected with SARS-CoV-2 is a key step for the control of the epidemic.At present,one of the standard method for COVID-19 diagnosis is viral nucleic acid detection by RT-PCR,but its widely application during the outbreak of pandemic was restricted due to its time-consuming procedure,complex operation,special requirements for instruments and facilities and so on.The National Health Commission released the "COVID-19 Diagnosis and Treatment Protocol(Trial Seventh Edition)" recommending the serological test as the diagnostic evidence of viral infection in patients with clinically suspected COVID-19 or convalescent disease and negative nucleic acid test.Serum antibody detection has been rapidly developed as an auxiliary detection method with advantages of simple operation,short detection time and easy assess to specimens.Different types of coronavirus may lead to the occurrence of cross-reaction of antibody detection,resulting into false positive the detection for SARSCoV-2.Objective: To evaluate the application value of antibody detection method in the diagnosis of SARS-CoV-2 infection and effect of antigenic cross reaction on SARS-CoV-2 antibody detection based on ELISA method,that provided theoretical guidance for the application of clinical serological detection.Methods: 1.Expression and purification of human coronavirus related proteins: The gene sequences of N,ECD,S1 or RBD proteins of seven human coronavirus(SARS-CoV-2、SARSCoV、MERS-CoV、HCoV-OC43、HCoV-HKU1、HCoV-NL63、HCoV-229E)were obtained from the NCBI database,and the corresponding proteins were designed,optimized,synthesized and expressed.2.Establishment and optimization of the ELISA method for detection of SARSCoV-2 antibodies: The ELISA was optimized with various coating protein and serum concentrations,blocking solutions and time,incubation time of primary serum antibody,ELISA antibody concentration and incubation time,and chromogenic time.In addition,the limit of detection was determined,and the reproducibility and stability of the experiments were investigated.3.Application of serum antibody detection for diagnosis of SARS-CoV-2: The N,ECD,S1 and RBD proteins of SARS-CoV-2 virus were used as coating antigen proteins,and the corresponding IgM and IgG antibodies of 85 serum specimens from 19 patients with COVID-19 were detected by ELISA method.The detection rate of COVID-19 by using different antigen proteins was analyzed,and the efficiency of antibody detection method in the diagnosis of SARS-CoV-2 infection was discussed.4.Effect of antigenic cross reaction on antibody detection of SARS-CoV-2-N: Serum IgM and IgG antibodies of N protein from 1792non-COVID-19 patients in our hospital were detected by ELISA method,and various causes of false positive antibodies in antibody detection were analyzed.Thereafter,the homology and evolutionary tree of the N proteins of the seven human coronavirus species were analyzed.Western blot was used to detect the antigenic cross reaction between different human coronavirus N proteins,and to investigate the influence of antigenic cross reaction on false positive antibody detection.Results: 1.Expression and purification of human coronavirus related proteins: After sequence optimization and protein expression and purification,the purified,soluble recombinant N proteins of 7 kinds of human coronavirus(SARS-CoV-2,SARS-CoV,MERS-CoV,HCoVOC43,HCoV-HKU1,HCoV-NL63,HCoV-229E)and the recombinant ECD,S1 and RBD proteins of SARS-CoV-2 were successfully prepared.2.Establishment and optimization of ELISA method for detection of SARS-CoV-2 antibody: The ELISA reaction was optimized as follows: coating 1 μg/mL protein overnight,sealed with 1%BSA for 120 min,followed by incubation for 90 min with 1:500 diluted serum,incubated with 1:500 diluted HRP-conjugated antibody for 90 min,and the color development time of 15 min.The limit of detection of SARSCoV-2 antibody by different ELISA methods: The cut-off values of detection of N,ECD,S1 and RBD proteins were 0.4,0.26,0.19 and 0.35,respectively.The result was designed as positive when the OD value exceeded the corresponding limit of detection and designed as negative otherwise.In addition,the ELISA method had good reproducibility and stability with the intrabatch and interbatch variation less than 15%.3.Application of antibody detection in diagnosis of SARS-CoV-2: Serum IgM and IgG antibody detection by ELISA can effectively distinguish COVID-19 patients from non-COVID-19 patients.Along with the increase of infectious period,the positive rate of serum immunoglobulin IgM and IgG from COVID-19 patients increased,and the positive rate of SARS-CoV-2-N IgG was greater than that of IgM.The positive rate of SARS-CoV-2-N IgG antibody was more than 90% after 15 days from the onset of infection,and the positive rate of N protein specific IgG antibody could even reach100% after 29 days.In addition,serum specific antibodies against N,ECD,S1,and RBD proteins in COVID-19 patients were continuously minitored,and serum IgM and IgG antibodies were simutaneously detected in all 19 patients(100%)over the invested period,while 2 patients showed relatively lower antibody titer to SARS-CoV-2 related antigens.4.Effect of antigenic cross reaction on antibody detection of SARS-CoV-2-N protein: Serum samples from 1792 non-COVID-19 patients were detected by ELISA for anti-N-IgM and antiN-IgG antibodies,and the false positive rate of was 2.5%(45/1792)and 0.3%(19/1792)for IgG and IgM,respectively.Endogenous(rheumatoid factor,heterotropic antibody,etc.)or exogenous(hemolysis,bacterial contamination,etc.)factors may lead to false positive.The cross-reaction between N protein antigens of different human coronavirus species is one of the reasons to cause false positive of serum antibody detection,and the degree of cross reaction was consistent with the results of homology analysis.Conclusion: Antibody testing can effectively distinguish COVID-19 patients from nonCOVID-19 patients.The detection of IgG for N proteins in COVID-19 patients showed the highest positive rate among the tested proteins,and the positive rate increased along with the infectious perioid,even reaching 100% at day 29 from the onset of infection.The continuous monitor of N,ECD,S1 and RBD specific IgM and IgG in serum of COVID-19 patients provided a basis for tracking the progression of COVID-19.However,it showed a high false positive by detecting the IgM and IgG with the antigen of recombinant SARS-CoV-2 N protein,accounting for the reason that there was a certain degree of cross-reaction between the N proteins of the seven human coronavirus,given that the degree of cross-reaction was related to the homology of the seven human coronavirus.Therefore,antibody testing should only be used as diagnostic evidence of viral infection in patients with clinically suspected COVID-19 or in patients with convalescent disease and negative nucleic acid tests. |
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