| Forest encephalitis,also known as Tick-borne Encephalitis（TBE）,is an acute zoonotic natural disease caused by the Tick-borne Encephalitis virus（TBEV）.It is characterized by lesions in the central nervous system,with a high mortality rate.The prognosis is that many patients still have neurological sequelae.So far,there are no effective treatment measures and antiviral drugs for forest encephalitis.Vaccination is the main means to prevent and reduce the incidence rate.Due to differences in individual age,immune response,and other factors after vaccination,vaccine ineffectiveness may result in breakthrough infection after exposure to the virus.Therefore,the detection of antibody levels after vaccination is an important indicator of vaccine evaluation.Currently,animal methods,Plaque Reduction Neutralization Test（PRNT）,and enzyme linked immunosorbent assay（ELISA）are commonly used for the detection of antibody titer levels.Animal methods and Plaque Reduction Neutralization Test are traditional methods,which have indispensable advantages such as intuition and sensitivity.However,they also require the manipulation of live viruses High requirements for the skills and professional level of operators,long test cycles,and other deficiencies;ELISA is based on antigen antibody specific reactions and uses the color of the chromogenic substrate as a criterion for evaluation.It has the advantages of simplicity,rapidity,specificity,and ease of standardization.It is widely used in various fields of biomedicine and vaccine evaluation.In this study,firstly,10 batches of inactivated forest encephalitis vaccine were randomly selected for immunization in mice from each batch of vaccine.At the same time,non immunized mice were used as the control group,with 30 mice in the test group and 30mice in the control group.On the 14th day after the initial immunization,the virus dilution in the test group was 10-2-10-6,and the virus dilution in the control group was 10-6-10-10.Each dilution was used to attack 6 mice,respectively,The immune protection index of 10batches of vaccine was calculated by observing and recording the survival rate of mice within 21 days.Before the intraperitoneal challenge,blood was collected from the orbit of the mouse,and the collected serum was isolated and inactivated,followed by a plaque reduction neutralization test.Before conducting the plaque reduction neutralization test,first determine the titer of the virus solution used in the test using the plaque method.Use the virus dilution used when the number of spots is 30 to 40 as the virus dilution used for PRNT.Set up both a cell control and a negative control in the test to determine the effectiveness of the test results and results.After incubating serum with different dilution levels with the virus solution,add it to the cells for virus adsorption,Based on the statistics of the number of plaque,the Reed Munch method was used to calculate the serum antibody titer.Indirect ELISA was used to detect antibody levels in the collected mouse serum from both the experimental and control groups.First,prepare a TBEV antigen specific detection enzyme-labeled plate,and conduct a semi quantitative determination of the titer of the TBEV stock solution used for subsequent coating.The TBEV antigen is coated on the enzyme-labeled plate through a capture method.Indirect ELISA is used to detect mouse serum with different dilution ratios.The average value and standard deviation of A450 nm in each group of negative mouse serum are tested to determine the detected Cut-off value,and the maximum serum dilution ratio determined as positive is used as the antibody titer of the sample.In summary,the animal method determines that the immune protection index of 10batches of vaccines is 1.0×107～1.5×108,all greater than 5×105,meeting the immunogenicity requirements of the inactivated forest encephalitis vaccine in the Chinese Pharmacopoeia（Part III）;The results of plaque reduction neutralization test on 10 groups of mouse serum showed that the average antibody titer was 2.5×103～2.5×104,the test results have a high correlation and consistency compared to the animal method,with a correlation coefficient of 0.97;The results of indirect ELISA for 10 groups of mouse serum showed that the average geometric titer of the antibody was 7.0×103～4.0×104,the test results also have a high correlation and consistency compared to the animal method,with a correlation coefficient of 0.98.The correlation and consistency of the ELISA test results are significantly higher than those of the plaque reduction neutralization test,which preliminarily indicates that the ELISA method is superior to the plaque reduction neutralization test in vaccine efficacy evaluation and antibody level detection,providing a foundation for the establishment of a new vaccine efficacy evaluation method. |
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