Establishment And Application Of Fluorescent Antibody Virus Neutralization Test In Hemorrhagic Fever With Renal Syndrome

Objective: To establish a laboratory standard method-FluorescentAntibody Virus Neutralization （FAVN） to detect the human serum levels ofneutralizing antibody of hemorrhagic fever with renal syndrome virus（HFRSV）. The method will be initially applied in Hebei province, toinvestigate the serum neutralizing antibody levels of HFRSV and theimmunological effect after the use of vaccine in healthy population so as toguide the prevention and control of HFRS.Methods:1Cell culture: according to the1:3points to pass, Vero-E6cells wereused to grow into monolayer, and then full digested with pancreatic enzyme,suspended in MEM medium. Then50μl was added in96-well cell cultureplate hole,37℃,5%CO₂incubator with an atmosphere of culture, until thecells grew into a single layer;2Dilution of virus solution: TCID50of the experimental strains wasfirstly determinated, then the virus solution was diluted to100TCID50/100μlspare;3Collection and dilution of the serum specimens: the serum samplessaved in the laboratory were as waiting serum both before and after the HFRSbivalent inactivated vaccination. The serum were diluted from1:2to1:64do a2-fold serial dilutions;4Neutralization process: the serum samples and virus-liquid were mixedin the37℃,5%CO₂cell incubator for1hour;5To Inoculate cells into the mixture: the Vero-E6cells that had growninto a monolayer cells were discarded the original medium, were added100μlof serum-virus mixture in each detection hole, and were cultivated in the37℃,5%CO₂cell incubator; 6Fluorochrome stain and microscopic observation: the96-well plate wasfixed with80%acetone, added fluorescent-labeled anti-HV monoclonalantibody that had been diluted to the working concentration, blowed dry, Thenobserved the infected cells under a microscope and recorded the results;7The detection of the sensitivity, specificity and stability of FAVN test:FAVN test and PRNT test were two ways to detect neutralizing antibodies inhuman serum before and after immunization with HFRS bivalent inactivatedvaccine for60share, compared the results of two methods to detect sensitivityand specificity of FAVN test;8The application of the FAVN test: according to the epidemic situationof the HFRS outbreak in Hebei Province, selected3high-risk counties ofHFRS as vaccine inoculation points, selected the healthy population from16to60year-old at each inoculation point, which were divided into four agegroups,20persons in each group, used stratified random sampling, andcollected the serum samples before and after vaccine inoculation of objects.The FAVN test was used to detect HFRS virus neutralizing antibody in thecollected serum.Results:1Different cell concentrations result in different fluorescent focus. onlywhen cell concentration was4×10⁴～4.5×10⁴/hole cell, microscope showedthat the cell distribution was uniform and neat, and that boundaries betweencells were clear, and that specific fluorescent particles could be observedclearly.2Serum-virus mixture in different temperatures resulted in differentfluorescent foci. To compare the serum-virus mixture under the condition of4℃overnight with the condition of at37℃,5%CO₂cell incubator for onehour, it turned out that the latter had bigger effect on virus titre than the former.So this laboratory carried out it under the condition of37℃and1hour forneutralization.3FAVN test had higher sensitivity. The FAVN test and PRNT test weretwo ways to detect neutralizing antibodies in human serum before and after immunization with HFRS bivalent inactivated vaccine for60share. ThroughFisher’s exact test, χ2=4.02, P<0.05, the difference was statistically significant.so it could be considered that positive detected rate of FAVN test was higherthan that of PRNT test.4FAVN test had higher specificity and stability. FAVN test was used todetect30vaccine human serums before immunization, and neutralizingantibody of30serums were negative, indicating that this method was specific.FAVN test was repeated three times for25test positive for serum samples,whose neutralizing antibody titre had been detected positive. The analysis ofvariance showed that there was no significant difference （F=3.08, P>0.05）,which indicated that this method was stable.5FAVN test was detected neutralizing antibody for the same populationof pre-immune and immune. A total of480serum samples in three countiesincluding Luannan, Beidaihe and Changli are detected, and the results showedthat there were7serum samples for neutralizing antibody-positive beforeimmunization, and for neutralizing antibody titer between10and20, andneutralizing antibody positive rate in human serums before immunization were2.92%; There were7people whose serums contained neutralizing antibody,and6people were farmers accounted for85.71%of the population of thelatent infection; serum neutralizing antibody after immunization risesobviously. Among240serum samples, there were169serum samples ofwhich the neutralizing antibodies after immunization were positive, andpositive rate of the antibody was mean70.42%. By the chi-square test, therewas a significant difference in positive rate of antibody before and afterimmunization （χ²=2.35, P<0.05）. Among169serum samples, there were104serum samples of which the ages were26to45, which suggested thatimmunological effect of vaccine was more better in young people.Conclusion:1Fluorescent Antibody Virus Neutralization （FAVN） test has beeninitially established to detect the human serum levels of neutralizing antibodyof hemorrhagic fever with renal syndrome virus. 2The sensitivity of FAVN test is higher than that of PRNT test, whichhas good specificity and stability.3Serum neutralizing antibody of HV before the vaccination are very lowin healthy population in Hebei province, People are generally susceptible toHFRS.4The immunological effects of HFRS bivalent inactivated vaccine aregood. the positive rate of serum neutralizing antibody after vaccination ishigher than70%in healthy population.