Rapid Detection Of Neutralizing Antibodies Against Rabies Virus Based On Competitive ELISA

Rabies caused by rabies virus(RABV)has a long history.The mortality rate of human and animal with clinical symptoms of rabies is 100%.Vaccination is considered to be the most effective method for the prevention and control of rabies,and the RABV neutralizing antibody titer ≥ 0.5 IU/mL in the serum after immunization is the standard of rabies vaccine recognized by the World Health Organization to achieve immune protection effect.At present,the main titration methods of neutralizing antibodies in rabies serum are Rapid Fluorescent Focus Inhibition Test(RFFIT)and Fluorescent antibody virus neutralization(FAVN),but the virus neutralization test is time-consuming,complicated,requires the operation of live RABV,and requires certain professional skills of operators,so it is not easy to be popularized and applied.Therefore,it is of great significance to develop a rapid and accurate method for the determination of neutralizing antibodies against RABV.RABV glycoprotein(G)is the main antigen that stimulates the production of RABV neutralizing antibody.There is a good correlation between the detection technology of RABV G specific antibody and virus neutralization test.In this study,semliki forest virusRABV G(SFV-RVG)and RABV G neutralizing monoclonal antibody were used as basic materials to establish a competitive ELISA(cELISA)method.In order to simplify and popularize the RABV neutralizing antibody determination method.The main research results are as follows:1.Inactivation mode of SFV-RVG.SFV-RVG has certain pathogenicity,and a high dose of SFV-RVG can kill 3-day-old suckling mice.It is required that infectious antigens should be inactivated before encapsulation.In this study,various methods including cELISA,indirect immunofluorescence assay and the mortality test of lactating mice were used to confirm that SFV-RVG could be completely inactivated by β-propiolactone at a ratio of 1:1000and rotating incubation at 4℃ for 12 h.2.Screening of optimal competing monoclonal antibodies.The cELISA method is established on the premise that the monoclonal antibodies used can distinguish the readings of positive and negative samples well.In this study,a monoclonal antibody was directly selected from 8 strains of RABV G monoclonal antibodies labeled with horseradish peroxidase(HRP)by cELISA to establish a cELISA method for RABV neutralizing antibody detection.Considering the specificity and sensitivity of cELISA method,strain 7A3 was determined as the best competitive monoclonal antibody.3.Self-built cELISA can quickly determine whether the titer of RABV neutralizing antibody in canine serum reaches 0.5 IU/mL.In this study,the detection system based on cELISA for rapid detection of RABV neutralizing antibody was assembled into kits after a series of optimization.The determination criteria of cELISA were determined by detecting 156 dog serum samples.ROC curve analysis results showed that: When the absorbance value of the sample well at630 nm/the absorbance value of the negative control well at 630 nm(S/N value)≤ 0.650,the results were determined as positive,that is,the RABV neutralizing antibody titer ≥ 0.5IU/mL.When S/N > 0.650,the result was negative,that is,the titer of RABV neutralizing antibody was < 0.5 IU/mL,suggesting that the body needs to be vaccinated with rabies vaccine to enhance immunity.The sensitivity,specificity,repeatability and coincidence rate of the kit were all good.In conclusion,the kit developed in this study is a safe,rapid,accurate and specific RABV neutralizing antibody qualitative detection kit,which is conducive to the scientific prevention and control of rabies based on antibody data.