| Objective:Using COVID-19 S protein as an envelope antigen,an indirect ELISA method was developed for testing the SARS-CoV-2 antibody.Furthermore,the diagnostic value of the indirect ELISA method for the detection of SARS-CoV-2antibody was evaluated and the feasibility of the method was assessed,so as to detect antibody produced in people who have received the COVID-19 vaccine or been infected with the COVID-19.Methods:Under the optimal conditions of the experiments for the optimal blocking solution,meanwhile maintaining the optimal ratio and time of the enzyme-labeled secondary antibody in the optimal conditions,and the optimal sample and color development solution determined by the COVID-19 antibody-positive sera,the optimal antigen concentration of SARS-CoV-2 S protein were determined by tessellation titration,and the optimal dilution ratio of COVID-19 antibody-positive sera was determined in the same method.And then,the critical values were calculated using the optical density values derived from the sera of 50 healthy controls.What is more,to understanding the the diagnostic value of the ELISA indirect method established in this experiment,the sera from different lung diseases,specimen traits and endogenous substances that may interfere with the interpretation of the results of this experiment were also tested.This study selected 85 healthy physical controls who were reexempted from inactivated COVID-19 vaccine,61 sera with COVID-19 antibodies by chemiluminescence as the experimental vaccine group.In addition,100 hospitalized patients with negative nucleic acid for COVID-19 and no abnormal pulmonary imaging were selected as the negative control group.By using S protein as the diagnostic antigen and conducting the ELISA indirect method,the sensitivity,specificity and positive predictive value,negative predictive value and diagnostic efficiency could be calculated after experiments,and the positive conversion rate could also be calculated after the injection of the COVID-19 vaccine.Results:As the result of our experiment,the optimal reaction concentration of S protein was determined by the tessellation titration method to be 6.4μg/m L,the optimal dilution ratio of serum as 1:100.In addition,the optimal closure conditions was found to be closed for 1 hour under 5%bovine serum albumin,and the optimal dilution of enzyme-labeled secondary antibody as 1:1000 when reacting for 1 hour.The optimal sample and chromogenic solution action time were found to be 45min and 30min,respectively,and Cut off value be 0.224.When testing for interfering factors,the false positive rate for specimens with different specimen traits were different,with 40%(8/20)for hemolysis,8.3%(2/24)for community-acquired pneumonia and 0%for other interfering factors among different categories of lung diseases.Meanwhile,theχ~2test was conducted,and drawn a conlusion that the hemolysed specimens could interfere with the interpretation of experimental results,while other interfering factors were not statistically significant.The result obtained from the analysis of the ELISA indirect method were set out,with the sensitivity to be be 85.2%(52/61)and specificity to be 89.0%(89/100).The positive and negative predictive values were calculated to be 82.5%and 90.8%,respectively,with a diagnostic efficiency of 87.6%and a positive conversion rate of 75.3%(64/85)after injection of the COVID-19 vaccine.Conclusion:Using S protein as the encapsulated antigen,the indirect ELISA method established in this study has a sensitivity of 85.2%and a specificity of 89.0%,which can be used to assist in the diagnosis of the antibody level in COVID-19 or previously infected patients and to evaluate the immune effect of COVID-19 vaccine. |
PDF Full Text Request