| The safety,effectiveness and quality stability of rabies vaccine are the key factors for the protection effect of this product.The release of vaccines from various manufacturers requires strict control of vaccine efficacy.The current detection of efficacy uses the NIH method.This method has the disadvantages of using animals,how to determine the end point of death,the need to manipulate live viruses,and the high coefficient of variation of the test resultsThere is a good correlation between the titer test(NIH method)and the ELISA detection of the antigen content in the vaccine.The research results show that the sandwich ELISA method is suitable for the in vitro titer test and has a tendency to replace the NIH methodAt present,the laboratory has a monoclonal antibody that recognizes the natural conformational GP,and has the conditions for establishing a double-antibody sandwich ELISA detection method for rabies virus GP antigen content and developing a detection kit.The following researches have been carried out in this paperEstablishment and optimization of ELISA methodPurpose A double-antibody sandwich ELISA method was developed for rapid detection of rabies virus(RabV)glycoprotein antigen content,and was validated and preliminary applied.Method A double-antibody sandwich ELISA was established with an anti-RabV glycoprotein genetically engineered antibody to detect the RabV glycoprotein antigen content.Verification of the specificity,linearity,accuracy,intra-plate and inter-plate precision and sensitivity of the established method,rabies vaccine produced by different manufacturers(strains are aG,PV,CTN,PM)and rabies at different stages of the process vaccine samples were tested for suitability.After the development of the rabies virus antigen detection kit is completed,the same person can operate the same microplate,different batches of microplates,and different people can operate the same microplate,and test the precision between the plates of the kit.Detect homemade rabies virus antigen detection kits in different batches after one month and four months to see whether the results have a good linear relationship to detect the homemade rabies virus antigen detection kits at 4? for a certain period of time after storage Verification of stability.Result The optimal dilutions of the capture antibody and the enzyme-labeled antibody were 1:2 000 and 1:4 000,respectively.This method does not cross-react with other human vaccines and excipient components.The linear range is between 0.0032 and 0.1031 IU/mL,and R2>0.98.The recovery rate for accuracy verification is between 97.0%and 110.1%.The coefficient of variation within the plate is<8.6%,and the coefficient of variation between plates is<13.9%.The rabies vaccines from different manufacturers and vaccine samples from different stages of the process showed good dose-dependent effects.The precision RSD value of the operation panel within the same person is less than 15%,the precision RSD of the operation panel between the same person is less than 15%,and the precision within and between the boards is qualified and good.The precision RSD value of the operating panel of different personnel is<15%.The test results of the homemade rabies virus antigen detection kit showed a good linear relationship whether it was tested after one month or four months.Conclusion A double-antibody sandwich ELISA method for the detection of glycoprotein antigen content in human rabies vaccine has been established,which meets the needs of quantitative detection.It can be used for rabies vaccine detection by aG,PV,CTN,PM and other different strains.It can also be used to detect samples in different process stages such as virus harvest solution,concentrated solution,inactivated solution and original solution.The homemade rabies virus antigen content detection kit is suitable for different operators,and can be stored at 4? for at least 4 months. |
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