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Study On The Method Of Detection Of Anti-poliovirus ? Sabin Strain Neutralizing Antibody Instead Of Live Virus

Posted on:2021-10-28Degree:MasterType:Thesis
Country:ChinaCandidate:B J ZhangFull Text:PDF
GTID:2480306308980789Subject:Biological products
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Poliovirus(PV)is the main pathogenic microorganism that causes paralysis in children under five years old.There are three wild poliovirus strains(serotypes 1,2,and 3).Since the successive application of the inactivated poliovirus vaccine(IPV)in 1955 and the Oral poliovirus vaccine(OPV)in 1963 and the global management and monitoring of poliovirus,the incidence of polio has been decreasing year by year.The World Health Organization(WHO)launched the Global Poliomyelitis Eradication Initiative(GPEI)in 1988.Following the announcement of the elimination of poliovirus type 2 wild strains in September 2015,it announced on October 24,2019 that the world had eliminated poliovirus type 3 wild strains,currently only poliovirus type 1 wild strains are circulating in Afghanistan and Pakistan.In addition,due to the use of OPV,there are occasional small-scale local occurrences of vaccine-associated paralytic poliomyelitis(VAPP)and vaccine-derived viruses(VDPV).The current global mission for poliovirus is to completely eradicate it.we must not only eliminate the currently circulating type 1 wild strains,but also eliminate VDPV and the long-lived polio attenuation discharged into the environment by infected patients.VDPV is mainly a kind of virus in which OPV is immunized in the human body due to immune deficiency or other reasons.The attenuated strain recovers its virulence and transmission and becomes pathogenic,including cVDPVs and iVDPVs,Against the background of global eradication of poliovirus,IPV will eventually replace OPV.At the same time,in the final stage of poliovirus eradication,the third edition of the World Health Organization Global Action Plan(GAP ?)pointed out that poliovirus needs to be operated in BSL-3 laboratories and only a few laboratories in the world can Save attenuated strains of polio virus.In order to meet the work requirements of general laboratories,there is an urgent need for new neutralizing antibody detection methods that can replace true viruses.This study is to construct a pseudovirus of poliovirus type 3 Sabin strains to detect the level of poliovirus antibodies in the population.First,with the poliovirus type 3 Sabin strains as a reference,a plasmid expressing poliovirus type 3 Sabin strains capsid protein P1 was constructed and combined with the T7 RNA polymerase and the P1 segment was replaced by luciferase.Virus backbone plasmid was co-transfected into 293T cells,and the pseudovirus of poliovirus type 3 Sabin strains,which has the ability to infect cells only once,was used as a detection tool instead of true virus.It has been determined that the diameter of the pseudovirus particle is about 30 nm and the shape is similar to the corresponding true virus,which can be used to evaluate the serum neutralizing antibody titer.At the same time,this study modified the ELISA method for detecting the D antigen of poliovirus type 3 Sabin strains,and improved the method for detecting the content of D antigen to detect anti-polio antibodies.First,coat bovine anti-poliovirus type 3 Sabin strains D antigen IgG,add D antigen after blocking with BSA,and then add rabbit anti-poliovirus type 3 Sabin strains D antigen IgG and human serum at the same time for competitive binding,and finally Adding goat anti-rabbit enzyme-labeled antibody,the modified method can be used as an alternative method to detect the titer of serum neutralizing antibody.This experiment confirmed that the enzyme-labeled antibody did not react with the serum,and also determined the optimal concentration of the antigen on the ELISA plate,and the dilution of the enzyme-labeled antibody was 1:1000.By referring to the results of the micro-neutralization test of the clinical serum poliovirus type ? Sabin strain,the serum with the higher neutralization titer was found as the sample for this study,and the standard curve of the OD value of R2=0.972 was obtained.Using this method to detect the neutralizing antibody titer of 8 sera from Kunming area,the positive rate of serum antibody and the traditional micro-neutralization test method were both 75%.There was no significant difference between the two methods.In summary,this experiment established a safe and reliable alternative to live virus to determine serum neutralizing antibodies through the construction of pseudovirus of the Sabin strain of poliovirus type ? and the improvement of the ELISA method.In the final stage of the eradication of poliovirus,the detection of serum antibodies in the population and the monitoring of the protection rate of the population against the Sabin strain of poliovirus type ? will prevent the virus from breaking out again.
Keywords/Search Tags:Poliomyelitis virus type ? Sabin strain, pseudovirus, ELISA antibody detection method, neutralization test, D antigen
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