| Chickenpox and herpes zoster are infectious diseases caused by varicella zoster virus（VZV）,which are prevalent all over the world.At present,the only effective and reliable means of prevention and control of the above diseases is to vaccinate corresponding vaccines,namely varicella live attenuated vaccine and herpes zoster vaccine（including herpes zoster live attenuated vaccine and recombinant herpes zoster vaccine）.Among them,Oka strain is used as the vaccine production strain for varicella live attenuated vaccine and herpes zoster live attenuated vaccine.The virus content of the live vaccine used to prevent herpes zoster is at least 14 times that of varicella live attenuated vaccine.The safety,effectiveness and quality controllability of vaccine products are the basis to ensure the immune effect of vaccine.Among them,vaccine titer,as the main index of effectiveness evaluation,is the key inspection item in the process of vaccine production and R＆D.For live virus,the efficacy of live attenuated varicella vaccine and herpes zoster vaccine is evaluated by virus titration results（i.e.virus titer）,and the legal verification method（included in Pharmacopoeia of the people’s Republic of China,VolumeⅢ（2020 Edition））is plaque method.This method has some disadvantages,such as long detection cycle,large human error,poor mobility,poor repeatability and so on.There are nine glycoproteins on the surface of VZV,including g E,g H,g L,etc,which are closely related to the pathogenicity and immunogenicity of the virus.ELISA detection can determine the number of virus particles with complete structure by quantifying the glycoproteins on the surface of VZV,so as to establish a good correlation with the detection results of plaque method.In view of the rapid,accurate and efficient quantitative detection of target protein,ELISA is a potential alternative to plaque method.In order to establish a double antibody sandwich ELISA method for rapid detection of VZV glycoprotein antigen content,firstly,the plaque method was optimized and verified in order to establish a corresponding relationship with the detection results of ELISA method.The optimization content includes the type of covering,the necessity of formaldehyde fixing steps,adsorption conditions and dyeing conditions,and the verification content includes repeatability,accuracy and precision.Secondly,a double antibody sandwich ELISA method was established with anti VZV glycoprotein genetic engineering antibody to detect the antigen content of VZV glycoprotein and establish a relationship with the results of plaque method;The optimization contents include antibody combination,antibody working concentration,coating conditions,sealing conditions,enzyme labeled antibody reaction conditions and color development conditions;The verification contents include specificity,linearity,accuracy and precision.The optimized plaque assay has high accuracy,repeatability and precision among personnel.It is verified that the ELISA method has good correlation with plaque assay,has good specificity for VZV glycoproteins and has no cross-reaction with other components;the linear range is 500～2600PFU/m L,R~2>0.95;the in-batch recovery rate is between 82.2%to 115.7%,and the coefficient of variation is<10.2%;the batch-to-batch recovery is between 80.5%to 118.6%,intraplate variation coefficient is<17.4%and inter-plate variation coefficient is<14.9%.In conclusion,a double-antibody sandwich ELISA method for the detection of VZV glycoprotein antigen was established in this paper.Thismethod can establish a relationship with the detection results of plaque assay,and the virus titer can be obtained indirectly by calculation.The method meets the needs of quantitative detection of VZV virus titer,and can be applied to the R＆D（Research and Development）and production process of live attenuated varicella vaccine and herpes zoster vaccine,so as to detect the virus titer of intermediate products and finished products quickly,efficiently and accurately. |
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