Establishment Of Fluorescence Immunosorbent Assay For Detection Of Rabies Virus G Protein Based On Quantum Dot Labeling

Rabies is caused by rabies virus,which can infect the central nervous system of almost all series of warm-blooded animals.Rabies is one of the acute infectious diseases with high mortality.Since there is no effective treatment for this disease,immunization with rabies vaccines should be done as soon as possible after being bitten by rabid animals.Among the structural proteins of rabies virus,glycoprotein is the only protein which induces the production of neutralizing antibodies.Therefore,to some extent,the content of glycoprotein can determine the protection effect of vaccine.Therefore,it is important to establish a rapid method to detect the content of rabies virus glycoprotein for the determination of vaccine immune efficacy.Despite Enzyme-linked immunosorbent assay(ELISA)is a widely used method for immunological detection,traditional ELISA methods still have shortcomings,such as relatively low sensitivity and high detection expenditure.Quantum dot is widely used in life sciences especially in vitro diagnosis due to excellent spectral characteristics and photochemical stability.In these applications,sensitivity of traditional ELISA can be significantly improved by combination with quantum dot.On the basis of ELISA detection and analysis,the monoclonal antibody(mAb)against rabies virus glycoprotein was used as capture antibody.Meanwhile,mAb labeled with quantum dot was used as the detection antibody.In this regard,it is of great significance for the determination of vaccine titer and immune dose to establish a Fluorescence-linked immunosorbent assay(FLISA)to quantify rabies virus glycoprotein.1.Preparation and identification of monoclonal antibody against rabies virus G proteinIn this study,three monoclonal antibodies against G protein of rabies virus were successfully obtained.They were named as 9F10,1F1 and 9G10,respectively.The characteristics of the three strains of monoclonal antibodies were identified.All the three strains of monoclonal antibodies were IgG antibodies,among which 9F10 and 1F1 were IgG2b and 9G10 were IgG2a.Their light chains were kappa.The results of chromosome detection showed that the number of chromosomes in 9F10 was 90.The number of chromosomes in 1F1 was 89.The number of chromosomes in 9G10 was 97.All three mabs can specifically identify the rabies virus lab-attenuated SAD,CVS-B2c,DRV-Mexico,DRV-HuNPN01,DRV-AH08,and SHBRV in indirect fluorescence immunity.By Western blot analysis,all three mabs could only identify SAD strain.The antigenic sites of 9G10were between 239-339 aa and the antigenic sites of 9F10 and 1F1 were between 1-139 aa.2.Establishment of FLISA method for detecting rabies virus G protein by quantum dot fluorescence immunosorbent assayThrough the quantum dot coupled antibody experiment,the results showed that the quantum dot modified antibody can normally stimulate fluorescence.The fluorescence wavelength did not be changed.The gel electrophoresis experiment showed that the quantum dot had been successfully modified on the antibody.Through pairing test,9F10was determined to be the capture antibody with the coating concentration of 2?g/mL.9G10 was coupled with ZnCdSe/ZnS quantum dots as the detection antibody with the dilution of 1:200.The reaction conditions were optimized with the coating concentration and antibody dilution showing above.The optimal blocking solution was 0.5%BSA+0.05%Tween-20 phosphate buffer.The blocking time was 1 h.The sample diluent was 1%BSA+0.05%Tween-20 phosphate buffer.The incubation time of the sample was 2 h.The incubation time of the quantum dot coupled antibody was 1 h.The sensitivity test results showed that the minimum detection limit of FLISA was 1:1600,and that of conventional double sandwich ELISA was 1:400.The standard curve,y=0.0002x+1.7,for quantitative detection was constructed based on the samples with gradient dilution.The R~2 was 0.9731.Specificity experiments showed that the method did not have non-specific reactions with canine distemper virus,canine parvovirus,293T cells,and BSR cells.In this research,3 monoclonal antibodies were successfully obtained.Fluorescence immune adsorption assay was established to detect the content of rabies virus glycoprotein based on the coupled quantum dots.It is a rapid and sensitive detection method for the determination of vaccine potency and immunizing dose in the future.