摘要
目的制备南定病毒（NDiV）标准血清，建立ELISA检测方法，为在人群中监测南定病毒感染状况提供方法。方法用南定病毒感染3周龄SPF级BALB／c小鼠，分别在0、7、14d以腹腔注射的方式免疫SPF级6-8周龄BALB／c小鼠，免疫血清用IFA法进行鉴定；南定病毒感染C6／36细胞，制备ELISA抗原，确定酶结合物、抗原和标准阳性血清的最伟工作浓度，建立南定病毒ELISA检测方法，进行敏感性、特异性、重复性和稳定性实验。结果制备南定病毒免疫血清，以IFA测定血清效价为1：320，与呼吸道合胞病毒、腺病毒均无交叉反应；建立了ELISA检测方法，确立了各种试剂的最佳工作浓度，其中酶结合物最佳工作浓度为1：10000，抗原为2．3μg／ml，阳性参考血清为1：200；ELISA检测灵敏度为1：3200。板内特异抗原、正常抗原平均变异系数为4．56％和5．17％，板间为3．38％和6．64％。稳定性实验相财偏差均小于20％。结论本研究制备的南定病毒免疫血清可作为血清学检测方法中的标准质控血清；建立的ELISA方法重复性、稳定性好，敏感性和特异性强，可用于南定病毒血清抗体检测。
Objective To prepare the standard serum against NDiV and establish a method of detecting NDiV antibody by ELISA so as to monitor the infection status of NDiV in population. Methods NDiV was cultivated by intracerebral inoculation in BALB/c mice aged 3 months, and immunogen was prepared. 6-8 weeks old BALB/c mice were immunized on days 0, 7 and 14 respectively by intraperitoneal injection of the immunogen. The harvested sera were verified for the sensitivity with IFA. C6/36 cells were infected with NDiV to prepare specific NDiV antigen, and ELISA antigen was prepared. The best working eoncentrations of enzyme conjugate, antigen and the positive serum were titrated. The NDiV ELISA detection technique was established, and the sensitivity, specificity, repeatability and stability were determined. Results The serum titer determined by IFA was 1：320, and there was no cross-reactivity with respiratory syncytial virus and adenovirus. The ELISA detection method was established. The best working concentrations of enzyme conjugate, antigen and positive reference serum were 1： 10,000, 2.3 μg/ml and 1： 200 respectively. The sensitivity of the ELISA detection was 1：3,200. The inter-assay coefficients of variation of specific antigen and normal antigen were 4. 56% and 5.17% respectively, and inter-plate coefficients of variation of specific antigen and normal antigen were 3.38% and 6.64% respectively. The stability test showed that the relative deviation was less than 20%. Conclusions The im- munized serum prepared in this study can be used as the standard control serum in NDiV serological detection methods. The established ELISA method has a good sensitivity, specificity, repeatability and stability, and therefore can be used in the serological detection of NDiV.
出处
《实用预防医学》
CAS
2016年第9期1144-1146,F0003,共4页
Practical Preventive Medicine
基金
广东省自然科学基金(2014A030313583)
