Development And Application Of A Blocking ELISA For RABA Antibody Detection Using The Glycoprotein Ectodomain As Coating Antigen

Rabies,a disease caused by rabies virus(RABV),is a highly lethal disease which can infect both human and warm-blooded animals.This disease causes around 60,000 deaths each year.It is incurable that once the clinical symptoms appear,the patient will die.However,it can be 100% prevented by appropriate post-exposure prophylaxis(PEP).As canine is the main reservoir host and transmitter,vaccination in dogs can effectively prevent the occurence and transmission of this disease.It is proved that 70% immunization coverage among canine can prevent the spread of rabies effectively.The viral neutralization antibody(VNA)level is a n indicator to evaluate the immunization effect and the immunization coverage.There are various methods to detect rabies antibody at present,among which the neutralization tests,in animals or in cells,are accepted internationally.The mouse neutralization test is not commonly used for it consumes much time and animals.The cell neutralization tests,including the Fluorescent antibody virus neutralization(FAVN)and Rapid fluorescent focus inhibition test(RFFIT),usually need 2~3 days to complete and require the cultivate of live virus and well trained technicians,making them more suitable for specialized laboratory.In China,laboratories at grass-root level usually use ELISA method to carry out the VNA antibody detection and surveillance of immunization coverage of RABV for limitation of the condition to conduct neutralization tests.The ELISA methods include indirect ELISA,competitive ELISA and blocking ELISA,and most of them use purified or partly purified RABV virus as coating antigen.The complicate composition of dog serum often arouse false results,hence the urgent problem to reduce false backgrounds in current RABV antibody-detection ELISA methods.The glycoprotein is one of the five structural proteins of RABV,and also an important one,with the ability to influence the virulence of RABV and to stimulate the viral neutralizing antibody to protect the body,which makes GP an ideal antigen for detection of anti-rabies antibody.GP is a transmembrane protein,making it difficult to get the whole protein from virus particles.It also requires high glycosylation ability of the expression system.The mature glycoprotein consists of three domains: the ectodomain,the transmembrane domain,and the cytoplasmic domain,while the ectodomain carries the antigen sites.Thus,in this study,the animal cell expression system was used to express the ectodomain of RABV glycoprotein,and a blocking ELISA,which was independent of the whole virus particle as antigen,was established to detect the RABV antibody using the expression product and an anti-GP monoclonal neutralizing antibody in our laboratory.This method was established trying to reduce the false results and provide a reliable method for immunization surveillance in large-scale samples.Methods and results are reported as follows:1.Establishment of stable ectodomain-expressing cell lines: The gene of the glycoprotein ectodomain of rabies virus BD06 strain was amplified,and was then inserted into the secretary vector pCMVSec to construct recombinant vector.The sensitivity of HEK293 cell against the screening drug zeocin was measured.The recombinant plasmids were transfected into the HEK293 cell with Lipofectamine 2000,then zeocin was used to select positive clones.The supernatant of these clones was identified for expression of the ectodomain by ELISA and Western.The cell lines were passaged continuously and were froze and thawed to evaluate the expressing stability.The results are as follows: the gene of the ectodomain,named G-e1317 was obtained and was successfully inserted into the expression vector pCMV-Sec,the recombinant plasmid pCMV-Ge was obtained.A 700?g/ml concentration of zeocin was appropriate for positive cell selection.After transfection,8 zeocin-resistant clones was obtained,5 of which showed good reactivity with the RABV monoclonal antibody 6F12 and the anti-his mAb in ELISA.In western blotting analysis,a strip about 60 kd appeared as expected when using anti-his mAb or anti-G mAb respectively.The expression level of the cell lines didn't influenced by passage or freeze.This showed that the cell lines obtained were stable relatively.2.The establishment of the blocking ELISA system for RV antibody detection: The ectodomain expressed by the cell line was used as antigen to develop the blocking ELISA,the conditions were optimized,including the selection of coating buffer,the optimization of coating time and temperature of the antigen,the selection of blocking buffer,the determination of concentration of the coating antigen and the dilution ratio of the mAb,the selection of the mAb,the optimization of reaction condition,the selection of washing buffer,the optimization of colorize condition and the using of stabilizer.25 RABV negative dog sera were tested,and the cutoff of the method was calculated according to the formula: The cutoff(PI value)= the mean PI value of the negative sera +2SD.The repeatability was evaluated by detecting a few serums by intra and inter assay.Finally,the optimized system was as follows: The antigen without coating buffer was coated for 1.5h at 37?,0.05%(v/v)PBST was used as washing buffer,after washing for three times,the wells were blocked by 20%(v/v)negative canine serum for 2h at 37?.After washing,serum samples were added for a 45-minute incubation at 37?.After the washing step,mAb 6F12 was diluted by 1:500 and was added for 1h reaction at 37?.Plates were finally washed and TMB substrate was added for a 7min colorization at 37?.The reaction was stopped by 2M sulfuric acid.The optical density(OD)value was measured.The accelerated testing result showed that there was no significant difference between the plates which were protected by the stabilizer and had been stored at 37? for 14 days and the new coated plates in detecting serum samples with different VNA titers,which proved the stabilizer showed good protection for the coated antigen.The cut-off was finally set at 32.1% according to the formula.All CV results obtained in both intra and inter assay were below 10%,which indicated this b ELISA established was repeatable and reproducible.3.The application of the RABV blocking ELISA method: Both FAVN and optimized ELISA method were used to detect 364 dog serum samples for rabies antibody.The ROC curve and the AUC were obtained by considering the FAVN test as a golden standard.The diagnostic sensitivity and specificity at the cutoff of 32.1% and the agreement between two tests were also obtained.Youden Index was used to evaluate the cutoff value.The results showed that for the 364 clinical samples tested,the diagnostic sensitivity of b ELISA was 88.9% while the specificity was 98.6%,the consistency between the two tests is 92.6%.The AUC was 0.964,which indicated this method was of great diagnostic value.According to the Youden Index evaluation,32.1% was a recommended cutoff.Thus it laid the foundation of the development of commercial ELISA kits.Conclusions are drawn through the research :1.In this study,stable cell lines were successfully established for expressing the ectodomain of RABV G protein,and the reactivity of the e xpressed product was proved by ELISA and WB,which laid the foundation of the application of the G ectodomain;2.A blocking ELISA for rabies antibody detection was developed using the expression product of the cell line and the mAb,the conditions were also optimized and finally established: Coat the antigen without coating buffer for 1.5h at 37?,use 20%(v/v)negative canine serum as blocking buffer for a 2-hour blocking at 37?,add serum samples for a 45-minute incubation at 37?,dilute the monoclonal antibody 6F12 by 1:500 and add it for a 1-hour reaction at 37?,0.05%(v/v)PBST is used as washing buffer for a three-time washing between each two steps above.After the antibody step,wash the p lates and add TMB substrate for a 7-minute colorization at 37?,stop the reaction and read the optical density(OD)value.3.The result of testing on clinical samples,including 226 positive samples and 138 negative samples,showed that the blocking ELISA had a high agreement with the FAVN test,which provided an important data to the development of commercial kits.Innovation of this research includes:1.It is the first time to express the ectodomain of the RABV glycoprotein using the animal cell expression system in a secretory way.The expression product in serum-free medium is directly used as coating antigen,which saves the cost of purification.Usage of the single component of glycoprotein as antigen decreases the background related to other components of the virus particle.2.It is the first time to use the glycoprotein ectodomain as antigen to develop a blocking ELISA for RABV antibody detection in our country,and consistency with the golden standard was validated.Kits were packaged in the laboratory.This method provides an effective tool for RABV immunizatio n surveillance,and is of practical significance for large scale RABV antibody detection.