Development Of Monoclonal Antibody Capture ELISA For Detection Of IgG Antibody To Japanese Encephalitis Virus

Japanese encephalitis(JE) is a zoonosis transmitted by mosquitoes,which pathogen is Japanese encephalitis virus(JEV).JEV causes human central nervous system changes with high mortality rate and severe sequela.JE is a vaccine-preventable disease.After vaccination or natural infection antibodies specific to JEV will occur and last for a long time.There are several methods to detect antibodies to JEV,such as neutralization test(NT),indirect immunofluoresence assay (IFA),hemaglutination inhibition test(HI),enzyme-linked immunosorbent assay (ELISA),and so on.NT is the golden standard for antibody detection but has strict requirement for technologist and has some limitation because of the live virus.IFA is specific and sensitive but has strict requirement for equipment.HI has low sensitivity. Compared with above,ELISA is an important method because of its specific, sensitivity,easy-done.At present,there is lack of good ELISA reagent to detect IgG antibody to JEV worldwide.Aim of this study is to construct an ELISA detection system which can rapidly detect JEV IgG antibodies and can be used to detect JEV IgG antibodies in the population.Details of the study and results are listed below:Development of monoclonal antibody capture ELISA for detection of IgG Antibody to Japanese Encephalitis Virus(GAC-ELISA for JEV)1.Establishment of serum panels for development of detection systemIn human bodies neutralization antibodies to JEV mainly exist at the type of IgG, so detection of neutralization antibodies can mainly reflect the level of IgG antibodies. In this study NT was carried out on 456 serum samples collected in 2006 and 80 serum samples were selected and put into serum panels.According to neutralization antibodies titers,20 were below 1:10,20 were 1:10-1:20,20 were 1:20-1:40,20 were above 1:40.2.Development of GAC-ELISA for JEV 2.1 Preparation of JEV antigenIn this study P3 strain was amplified in C6/36 cell lines.After routine treatment, virus supernatant was inactivated.The inactivated supernatant was inoculated on C6/36 cell lines and no CPE was observed for 3 sequenced passages which indicated that the virus was fully inactivated.In order to improve the concentration and purification,300mL inactivated supernatant was disposed using Millipore Labscale system and 12mL concentrate was obtained.The filtrate and concentrate was tested with molecular biological method and ELISA.The results showed that the amplification of PrM segment was found in the 10~4 times diluents of concentrate but wasn't found in the filtrate and its diluents.ELISA results showed that the antigen was condensed by 20 to 30 times but no antigen was found in the filtrate.2.2 Preparation of JEV monoclonal antibody(MAb)JEV07# was resuscitates.Supernatant was collected 3 days later to test whether it could produce MAb or not.It indicated that JEV07# could produce Mab from the positive ELISA result and positive IFA result.While enough JEV07# cells were obtained the supernatant was tested again,it showed the OD value of was 1.397. JEV07# cells were injected into 10 Balb/c mice.25mL ascites were obtained.SP2/0 cells were injected into 1 Balb/c mouse and 1mL ascites were obtained.ELISA results showed the titer of ascites from JEV07# was higher than 8×10~3 while ascites from SP2/0 were negative.2.3 Development of GAC-ELISA for JEVIn this study specific and homogeneous JEV MAb was used to capture JEV antigen in order to purify it and lower problems triggered by serum protein and cell components.Determination of working concentration:The plate was coated with sufficient human IgG,HRP conjugated anti-human IgG was diluted and dilution 1:80,000(at 1.0 of OD value) was determined to be the working concentration.MAb was diluted and coated with coating buffer,treated antigen was diluted with wash buffer and added to the plate,then serum and HRP conjugated anti-human IgG was added with 1:100 dilution and 1:80,000 dilutions respectively.The results showed that 1:2,000 MAb, 1:100 antigen,1:100 serum and 1:80,000 HRP conjugated anti-human IgG was the best working dilution.At these working dilutions max P/N(23) was obtained and the OD value produced by negative control is below 0.1.Valuation of sensitivity:9 different-OD-value positive samples were detected by GAC-ELISA.Weakly positive samples(OD value≤0.4) were positive at dilution of 1:400,positive samples(0.40.8) was at the highest dilution of 1:12,800.These results showed sensitivity of the way was high.Valuation of stability:9 different-OD-value positive samples and 3 negative samples were detected repeatedly.Each sample was detected four times.The positive are still positive and the negative are still negative.CV of each sample was less than 6.8%.These results showed that the way had good stability.Evaluation of GAC-ELISA for JEVTo further evaluate the effect of GAC-ELISA,GAC-ELISA,national indirect kit and import kit compared using IFA as the standard.And some discussion of the relationship between ELISA and NT was done.Performance evaluation of GAC-ELISA,national indirect kit and import kit:Reliability evaluation of three different IgG ELISA methods showed that GAC-ELISA and import kit had higher reliability(97.5%)than national indirect kit(93.8%);Taking IFA as the standard,performance evaluation showed that GAC-ELISA has higher consistency(91.3%) and higher sensitivity(95.6%) than other two kits and good specificity(85.7%);import kit has 100%specificity but low sensitivity;both specificity and sensitivity of national indirect kit are lower than 80%.The relationship between ELISA and NT:Analysis of the consistency between ELISA and NT showed that GAC-ELISA had higher consistency(80%) than other two kits;Scatter chart showed there was no linearity between the results GAC-ELISA and NT.In summary,this study GAC-ELISA was developed using JEV MAb.Performance evaluation showed that the new GAC-ELISA had high consistency(91.3%) with IFA and higher sensitivity(95.6%) and specificity(85.7%) than other two kits. GAC-ELISA can be used as a routine method to detect IgG antibodies to JEV in population.