| Rabies is a highly fatal and zoonotic infectious disease caused by the neurotropic rabies virus(RABV),timely immunization of rabies vaccine is the only strategy for prevention of rabies.At present,the most reliable way to determine the levels of anti-RABV antibody after vaccination is whether can protect against rabies.Antibody detection methods require RABV positive and negative sera as reference materials for experimental control and result determination.There are no rabies virus antibody positive and negative sera reference materials that can meet the technical requirements of both international and domestic standard materials.In this study,Combined with the World Organization for Animal Health(OIE)standard serum preparation technology,Chinese Veterinary Pharmacopoeia and the National Metrology Technical Specification JJF1343-2012,the preparation process of the rabies virus antibody serum standard substance was formulated.The healthy Kunming dog hyperimmune serum and unimmunized negative serum prepared in the laboratory as raw materials.Sera were inactivated at 56℃ followed by filtration,aliquoting and titration of neutralizing antibodies by FAVN.After the screening is qualified,it is deternined reference sera candidates.Positive serum(108 bottles,1m L/bottle)and negative serum were lyophilized(100 bottles,1m L/bottle).Perform all tests for reference materials according to the preparation procedure.The results showed that the vacuum degree of certified reference materials was qualified and the residual moisture content meets the requirements.There was no mycoplasma contamination and rabies virus;The uniformity of lyophilized serum was good.the sera were stable by 56 days at both 4°C and 25°C,stable storage at 37℃ for 14 days,56℃ for 7 days.Stable at least six months at-20°C and-80°C.The certified values of the postive and negtive reference sera were 4.46±1.49 IU/m L and 0.02 IU/m L respectively.The successful preparation of rabies virus antibody serum reference substance candidate laid a foundation for the application of national certified reference substance in the next step.With the developed reference sera,using the “gold standard” for titrating rabies neutralizing antibody recommended by the World Health Organization(WHO)and the World Organization for Animal Health(OIE).More than 1,000 sera of dogs stored in laboratory were tested for neutralizing antibody.190 dog serum samples were screened out to establish a serum bank.Use this serum bank to evaluate 5 commercially available ELISA test kits.The results showed that the overall coincidence rate of 5 kits with FAVN was 68.95%～ 77.89%,the sensitivity of these kits was 52.63%～98.25% and the specificity was 40.79%～93.42%.ELISA kits are mainly used for vaccine immunity evaluation or dog immunity coverage screening.If the test is used for immune coverage screening,it is recommended to choose kits B and C that are more consistent with the gold standard FAVN.If the test purpose is to boost immunization of animals whose antibody levels are not up to standard,kit A with better specificity is recommended.However,the detection results of the five kits and the FAVN method are significantly different,that is,the ELISA kits cannot replace FAVN for the identification of a single serum sample,and can only be used for the preliminary screening of antibody levels.Through a comprehensive comparison of commercially available rabies virus antibody ELISA test kits,it is proposed to choose the appropriate ELISA kit according to different testing purposes.This method can greatly improve the effectiveness of antibody monitoring and is useful for evaluating the effectiveness of vaccines or immunizing dogs,coverage screening provides evidence. |
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