| Background and ObjectivesThe Novel Coronavirus(2019-nCoV),with its strong transmissibility and pathogenicity,has now spread throughout the world,causing 200 million infections and 4 million people deaths,putting enormous pressure on global medical resources and economic development.Vaccination is an effective means of preventing2019-nCoV pandemic.At present,there are several approved vaccines available in China and abroad,including BioNTech/ Pfizer and Modena RNA vaccines,Astrazeneca and Concino adenovirus vector vaccines,sinovac and Sinopsin inactivated vaccines.According to public data,the effective rates of various vaccines are 95%,94.1%,70% and 79% respectively.The price of inactivated vaccines is relatively moderate and only 2-8℃ is needed for transportation,which is conducive to their universal application in non-developed countries.2019-nCoV inactivated vaccines have been approved for emergency use in China,THE United Arab Emirates and Bahrain.Bahrain and Egypt.After vaccine immunization,the plasma cells can secrete immunoglobulin,part of the immunoglobulin can combine with pathogens,blocking the pathogen infection,this part of the immunoglobulin is neutralizing antibodies,usually the IgG antibody includes neutralizing antibody.In theory,only neutralizing antibody levels can evaluate the level of protection of the body after infection or vaccination.Recently patient follow-up study carried out by professor Cao Bin and Zhang Dingyu show that the antibody positive rate decreased from 96.2% to 58.5% after 6 months discharge,and the average antibody titer decreased from 19 to 10.But the general population neutralizing antibody change after vaccination is not known,so studies are needed to study the patterns of neutralizing antibody production and change in the general population after vaccination.The gold standard for neutralizing antibody detection is live virus neutralization test,but the experimental needs to be operated in P3 laboratory,which is not suitable for large-scale vaccination.Therefore,a neutralizing antibody detection method with convenient and high throughput is needed.This research based magnetic particles chemiluminescence technology platform,try to establish a high-throughput,rapid,automatic neutralizing antibody detection method,the sensitivity,specificity,repeatability,stability were evaluated to meet the needs of large-scale standardized detection.The established method was used to monitor the change rule of neutralizing antibody after vaccination,providing data support for strengthening immunization.Methods1.Preparation of magnetic particle suspensionCarboxyl surface magnetic particles were activated by EDC and NHS to form active intermediates.Then,the recombined homologous dimer ACE2 protein(Fc label)was added,and the ACE2 protein was fixed to the magnetic particle surface by chemical linking method.Then,preservation solution containing protein and polyhydroxyl compound was added.2.Preparation of enzyme conjugatesThe horseradish peroxidase was oxidized by sodium periodate to form aldehyde group,then the recombined RBD protein was added,and conjugated with horseradish peroxidase by chemical linking method,and then diluted in a diluent containing protein and surfactant.3.Establishment of reaction systemThe dilution ratio of samples,the amount and sequence of adding samples,sample diluents,magnetic particle suspensions,enzyme conjugate,and reaction time were selected,so that neutralizing antibodies and ACE2 protein coated on magnetic particles in the samples could fully compete with RBD protein.4.Establishment of reference intervalSelect samples from healthy and vaccinated populations.The neutralizing antibody titer greater than or equal to 1:4 by live virus were considered positive.The reference interval is determined by Receiver Operating Characteristic Curve,that is,the sensitivity and specificity of detection under different critical concentration values are assumed.The maximum point of the Youden index was taken as the critical reference value.5.Method performance evaluationSensitivity: Live virus neutralization test is the gold standard,and the antibody titer of live virus neutralization greater than or equal to 1:4 were considered positive.Sensitivity = positive number of this method/positive number of live virus neutralization test ×100%.Cross-reactivity: Verifies cross-reactivity with antibodies to the following pathogens,including influenza A virus,influenza B virus,endemic human coronavirus,rubella virus,respiratory syncytial virus,enterovirus,adenovirus,Chlamydia pneumoniae,mycoplasma pneumoniae,measles virus,and mumps virus.Specificity: To detect samples from different populations before December 2019,including children with respiratory tract infection,adults with respiratory tract infection,patients with hypertension,patients with myocardial inflammation,and people with normal physical examination.Specificity = number of negative samples detected by this kit/total number of samples ×100%.Precision: 5 samples,including negative samples,critical positive samples,positive samples.Each sample was 3 repeated tested in the morning and afternoon every day for 4 consecutive days.A total of 24 data were obtained from each sample.The mean and standard deviation of 24 data of positive and weak positive samples were calculated respectively,and the coefficient of precision variation = standard deviation/mean ×100%.6.Neutralizing antibody surveillance in novel Coronavirus patients2019-nCoV infected person,including mild,ordinary and severe cases.Serum samples were collected at acute stage(within 2 weeks after onset)and at recovery period of 1 month,3 months and 6 months,respectively.The concentration of neutralizing antibody was detected with this method,and the change trend of neutralizing antibody concentration over time was monitored.7.Neutralizing antibody monitoring after immunization with inactivated vaccineSerum samples were collected at 1,2,3 and 4 weeks after the first dose of novel coronavirus vaccine,and at 1,2,3,4,6,10 and 16 weeks after the second dose of coronavirus vaccine.The concentration of neutralizing antibody was detected with this method,and the change trend of neutralizing antibody concentration over time was monitored.Results1.Magnetic particle suspension was prepared by coupling carboxyl surface magnetic particles with ACE2 protein expressed in HEK293 cells.ACE2 protein is a homologous dimer with human Fc label,with molecular weight of 127.3KD and purity of 86.5%.The activation buffer of ACE2 protein coupling magnetic particles was acidic buffer with PH value of 4.7-5.0,and the activation time was 1 hour.The coupling buffer was PBS buffer with neutral PH and the coupling time was 2 hours.Protein conjugation was 0.04μg/ test.Adding 1%BSA to BIS-Tr IS buffer can protect ACE2 protein activity,while adding 5% glycerol +5% sucrose can improve the freeze-thaw stability of magnetic particle components.2.The RBD protein(319AA-541AA)expressed in HEK293 cells was coupled with horseradish peroxidase to prepare the enzyme conjugate.The RBD protein C terminal had 6 His tags,the molecular weight was 32.8KD,and the purity was 96.5%.The optimal coupling conditions of RBD protein and horseradish peroxidase were oxidized with sodium periodate for 20 minutes.Coupling mole ratio 1:2;Reaction time 16 hours.The stability of the enzyme binding was the best in the bis-Tr IS buffer with 1%BSA,0.2%Casein and 0.1% Tween 20,and the luminescence value decreased by about 15%.3.Sample dilution selected Tris-Na Cl buffer with 1%Casein and preservatives.Rabbit anti-RBD polyclonal antibody was selected as the calibrator and diluted in human serum to prepare with minimal matrix effect.Calibration is traceable to2019-nCoV WHO Standard 20-136.4.The one-step competitive method was selected.20 ul sample,20 ul magnetic particles,50 ul sample diluent,50 ul enzyme conjugate were added and react,and the results could be get in 17 minutes.5.The critical concentration is 22.3IU/ m L based on Youden index.6.The sensitivity of the kit was 93.78% with 30IU/ m L as the critical value,and96.27% with 20IU/ m L as the critical value.Specificity is greater than 99.5%;Coefficient of precision variation<10%.7.In 2019-nCoV infection patients,it was found that the neutralizing antibody reached its peak at the first month of infection and gradually decreased thereafter.From the first month to the sixth month of infection,the titer of neutralizing antibody decreased by an average of 4-5 times.The level of neutralizing antibody in mild patients was significantly lower than that in normal and severe patients.8.The positive rate of neutralizing antibody continued to increase after immunization with vaccine,above 98% 3-4 weeks after two doses of vaccine.In most individuals,the antibody titer reached the peak at 3-4 weeks after the second doses,and decreased significantly at 3-4 months,and less than 30% at 6 months.ConclusionsBased on the principle that neutralizing antibody can block the RBD protein bind to human cell receptor ACE2,we established a 2019-nCoV neutralizing antibody detection method on the magnetic particle chemiluminescence technology platform.This is an automatic method,put on the sample to be tested,in 20 minutes it can report the neutralizing antibody concentration results,and 200 samples results per hour.This method has a high coincidence rate with live virus neutralization test,and can be used as a substitute method for live virus neutralization test to detect neutralizing antibodies quickly and efficiently.The changes of neutralizing antibodies in 2019-nCoV infected persons and inactivated vaccine vaccinated persons were regularly monitored by this method.It was found that neutralizing antibodies reached their peak one month after infection or two-dose immunization,and the antibody positive rate was over 98%,then began to decline.After 6 months,the titer of neutralizing antibodies decreased by 4-5 times on average,and the positive rate was less than 30%.It indicates the necessity of strengthening immunity. |
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