猪丹毒灭活疫苗效力检验血清学方法建立

Establishment of Serological Method for Efficacy Test of Inactivated Swine Erysipelas Vaccine

关键词:  猪丹毒  疫苗  效力检验  竞争ELISA

Key words:  Erysipelothrix rhuriopathiae  vaccine efficacy test  competitive ELISA

摘要:

建立了猪丹毒丝菌抗体竞争ELISA检测方法，用于猪丹毒疫苗效力检验。用SpaA蛋白(纯度90%)和辣根过氧化物酶(HRP)标记的SpaA蛋白单克隆抗体，建立猪丹毒丝菌竞争ELISA抗体检测方法，确定检测结果的判定标准，并对该方法的特异性、敏感性、重复性(批间重复性和批内重复性)等进行评价。建立效力检验血清学方法与免疫攻毒法的平行关系，并对两种方法符合率进行评价。结果表明，SpaA的最佳包被浓度为2.0 ng/μL，阴阳性对照血清的最佳稀释倍数为1∶75，HRP标记的单克隆抗体的最佳工作浓度为0.2 ng/μL。ELISA结果判定标准为血清样本抑制率PI≥8.0%时判定为阳性，PI＜8.0%判定为阴性。该方法特异性良好，与空白小鼠阴性血清及A型和B型猪多杀性巴氏杆菌阳性血清均不发生交叉反应；敏感性良好，与敏感性血清样品可呈现不同梯度抑制率；重复性良好，批内重复和批间重复性试验结果变异系数均小于10%；与效力检验免疫攻毒法具备平行关系，小鼠血清效价≥1∶100时可抵抗1000 MLD强毒攻击；对用不同冻干代次菌种制备的4批猪丹毒丝菌灭活抗原和来自4家企业的7批猪丹毒灭活疫苗进行检测并与免疫攻毒实验结果比较，总符合率为100%。建立的猪丹毒丝菌抗体竞争ELISA检测方法可以作为猪丹毒疫苗的效力检验方法。

Abstract:

To establish a competitive ELISA method for detection of antibodies against Erysipelothrix rhuriopathiae for the efficacy test of swine erysipelas vaccine. The SpaA protein (purity 90%) and monoclonal antibody against SpaA protein labeled with horseradish peroxidase (HRP) were used to establish a competitive ELISA antibody detection method for Erysipelothrix rhuriopathiae. To determine the criteria for detection results, the specificity, sensitivity, and repeatability (inter batch repeatability and intra batch repeatability) of the method were evaluated. Establish the parallel relationship between the serological method of efficacy test and the immune challenge method, and the coincidence rate of the two methods was evaluated. The results showed that the optimum coating concentration of SpaA was 2.0 ng/μL, the optimal dilution ratio of the negative and positive control serum was 1∶75, and the optimal working concentration of HRP labeled monoclonal antibody was 0.2 ng/μL. The ELISA results were determined as positive when the inhibition rate of serum samples PI≥8.0%, and negative when PI<8.0%. The specificity results indicated this method did not cross react with the negative serum and the positive serum of type A and B Pasteurella multocida derived from pigs; The sensitivity was qualified and showed different gradient inhibition rates employed with the sensitive serum samples; The repeatability was good, intra and inter batch repeatability test results showed the coefficient of variation was less than 10%; It had a parallel relationship with the potency test immune challenge method, when the mouse serum titer was ≥1∶100, it could resist 1000 MLD virulent challenge; Four batches of inactivated antigens of Erysipelothrix rhuriopathiae prepared by different freeze-dried strains and seven batches of inactivated swine erysipelas vaccines from four enterprises were tested and compared with the results of immune challenge test, the total coincidence rate was 100%. The established competitive ELISA method for detection of antibodies against Erysipelothrix rhuriopathiae can be used to test the efficacy of swine erysipelas vaccine.