Hu Zhe, Chang Hao, Ge Man, Lin Yuezhi, Wang Xuefeng, Guo Wei, Wang Xiaojun
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, CAAS, 427 Maduan Street, Harbin, 150001, People's Republic of China.
Appl Microbiol Biotechnol. 2014 Nov;98(21):9073-81. doi: 10.1007/s00253-014-6078-8. Epub 2014 Sep 26.
An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was established based on two monoclonal antibodies (mAbs) for the quantification of equine infectious anemia virus (EIAV). Two p26-specific monoclonal antibodies were developed in mice. The mAb 9H8 was coated in microtiter plates as the capture antibody; the other mAb, 1G11, was coupled to horseradish peroxidase (HRP) and used as the detection antibody. The limit of detection for the EIAV p26 protein was 0.98 ng/ml, and the linearity range was 3.9-62.5 ng/ml. The sensitivity of p26 AC-ELISA for the detection of the virus (EIAV infectious clone, FDDVcmv3-8) was the same as that for the purified p26 protein. No cross-reaction with other equine viruses was observed by this method. The intra- and inter-assay coefficients of variation were below 8.3 and 10.3 % for testing p26 and FDDVcmv3-8, respectively. The AC-ELISA was also compared to Western blotting (WB) and reverse transcriptase (RT) assays, validating the sensitivity, accuracy, and reliability of this method. Both the AC-ELISA and RT assay showed good agreement, with a correlation coefficient of R (2) =0.9946. Sample analysis showed that this AC-ELISA is a useful tool for quantifying EIAV p26 in cell lysates and culture medium.
基于两种单克隆抗体（mAb）建立了一种抗原捕获酶联免疫吸附测定（AC-ELISA），用于定量检测马传染性贫血病毒（EIAV）。在小鼠中制备了两种针对p26的单克隆抗体。将单克隆抗体9H8包被在微量滴定板上作为捕获抗体；另一种单克隆抗体1G11与辣根过氧化物酶（HRP）偶联，用作检测抗体。EIAV p26蛋白的检测限为0.98 ng/ml，线性范围为3.9 - 62.5 ng/ml。p26 AC-ELISA检测病毒（EIAV感染性克隆，FDDVcmv3-8）的灵敏度与检测纯化p26蛋白的灵敏度相同。该方法未观察到与其他马病毒的交叉反应。检测p26和FDDVcmv3-8时，批内和批间变异系数分别低于8.3%和10.3%。还将AC-ELISA与蛋白质印迹法（WB）和逆转录酶（RT）测定法进行了比较，验证了该方法的灵敏度、准确性和可靠性。AC-ELISA和RT测定法显示出良好的一致性，相关系数R(2)=0.9946。样本分析表明，这种AC-ELISA是定量检测细胞裂解物和培养基中EIAV p26的有用工具。
