The Development Of Indirect Hemagglutination Test For The Detection Of EBOV And Its Application In Antigen And Antibody

Ebolavirus disease(EVD)is an acute and potent infectious disease caused by Ebola virus(EBOV)with high morbidity and mortality in humans and primates.The disease was first discovered in the Democratic Republic of Congo(formerly Zaire)in 1976,and broke out in West Africa in March 2014.It has spread in Liberia,Guinea,Nigeria,Senegal,Sierra Leone and other countries.The death of this epidemic and the number of people who died in the 40 years since the first outbreak in 1976 were identified as "the strongest in history." According to the World Health Organization(WHO),by June 10 th,2016,there were 28,616 cases of globally diagnosed and suspected infections,including 11,310 deaths.In 2018,the Ebola outbreak was once again prevalent in the Central African countries.In the Central African countries,the Congolese outbreak was widespread.By March 5th,2019,the Ebola outbreak in the 10 th round of the Democratic Republic of the Congo reported 907 cases,of which 569 died.Due to the high morbidity and high mortality of the disease,serious economic losses and casualties were caused to the affected countries and affected areas.Although there are no reports of cases of EBOV infection in China,it poses a serious potential threat to China with the frequent international trade and the globalization of economic trade.At present,there are still no effective prevention and specific treatment drugs for EVD both in domestic and abroad.Therefore,in order to prevent the invasion of the disease,we must strengthen the detection of the entry and exit of the disease,and urgently need to establish a simple,rapid and specific detection method for EBOV antigens and antibodies.To this end,this study established EBOV antibody positive indirect hemagglutination test method and EBOV reverse indirect hemagglutination antigen test method,in order to provide technical reserve for EVD epidemic monitoring and EBOV antigen and antibody detection,in case of emergency.1.Development and preliminary application of EBOV positive indirect hemagglutination antibody detection method.Using purified EBOV glycoprotein(GP)as antigen to sensitize hydroformylated sheep erythrocytes and establish EBOV positive indirect hemagglutination antibody detection method.When the concentration of antigen-sensitized red blood cells is 160 μg/mL,the optimal reaction conditions for the method are 1 h at 4 °C,and the method can specifically detect EBOV antibodies,and Marburg virus and West Nile virus.Positive serum such as Rift Valley fever virus did not react with it.Using this method,20 samples of EBOV hyperimmune horse serum,purified IgG and purified immunoglobulin F(ab’)2 were detected,and the horse serum of EBOV VLPs vaccine harvested at different immunization times was detected,and the test results were obtained.Compare with the results of the pseudovirus neutralization test.The results showed that the method was consistent with the overall trend of the detection results of the pseudovirus neutralization test.In summary,this study successfully established a positive indirect hemagglutination test for EBOV antibodies.This method requires no special instruments and equipment,and is safe,simple and fast,and provides another new test for the evaluation of the efficacy of EBOV vaccine after immunization method.2.Development and preliminary application of EBOV reverse indirect hemagglutination antigen detection method.In this study,EBOV virus-like particles(EBOV VLPs)were used to immunize horses with positive serum.First,IgG was purified from the serum of the primary serum by saturated ammonium sulfate precipitation,and then purified by Protien G column affinity chromatography.The IgG is sensitized to the glutaraldehyded sheep erythrocytes,a reverse indirect hemagglutination antigen assay is established,and the optimal reaction conditions are optimized for the method.When the concentration of antibody-sensitized erythrocytes is 40 μg/mL,at 37°C 1 h is the optimal reaction condition of the method under the condition.This method was employed to detect EBOV pseudoviruses with known virus titers prepared in different batches.The results showed that the EBOV pseudovirus titer was consistent with the overall trend of hemagglutination results,that is the virus titer of the pseudovirus was high,and the relative hemagglutination test was high;on the contrary,when the pseudovirus titer was low,the corresponding hemagglutination test the result is low.This study successfully established EBOV reverse indirect hemagglutination antigen detection method,which is simple,rapid and does not require special equipment,can report the test results within 1 h,be operated in the ordinary laboratory and be used for EVD antigen The rapid detection has certain practical application value for monitoring and rapid diagnosis of EVD diseases.