Fray M D, Mann G E, Charleston B
Institute for Animal Health, Compton, Berkshire, RG20 7NN, Newbury, UK.
J Immunol Methods. 2001 Mar 1;249(1-2):235-44. doi: 10.1016/s0022-1759(00)00359-8.
We describe here a specific and sensitive assay for biologically active bovine type-I interferon (IFN) in an Mx/CAT reporter gene assay. The assay is based on Madin-Darby Bovine Kidney cells transfected with a plasmid, containing a human MxA promoter driving a chloramphenicol acetyltransferase (CAT) cDNA. CAT expression was quantified in a commercially available enzyme linked immunosorbant assay. The response to recombinant bovine INF-alpha(1) was dose dependent between 0.25 and 125.0 iu/ml and was shown to be specific for type-I IFN as no significant effect was seen with a number of other cytokines, including IFN-gamma. This Mx/CAT reporter assay also has advantages in terms of simplicity and reliability over conventional cytopathic effect reduction assays used to quantify the IFN activity in bovine samples. The Mx/CAT reporter assay was used successfully to measure trophoblast derived type-1 IFN activity (IFN-tau) in uterine flushings collected from pregnant cows. IFN-tau is the pregnancy recognition signal produced in ruminants by pre-implantation embryos and was shown to increase markedly between the 12th (0.7+/-0.14 iu/ml) and 18th (44085.0+/-14414.2 iu/ml) day of pregnancy. In contrast, IFN-tau activity remained basal (0.5-0.7 iu/ml) in inseminated non-pregnant animals. Duplicate samples analysed using a cytopathic effect reduction assay correlated well (P<0.001; r(2)=0.945) with IFN levels obtained using the Mx/CAT reporter assay, confirming the reporter assay as a reliable substitute for the standard anti-viral IFN assay.
我们在此描述一种在Mx/CAT报告基因检测中用于检测具有生物活性的牛I型干扰素（IFN）的特异且灵敏的检测方法。该检测方法基于用一种质粒转染的Madin-Darby牛肾细胞，该质粒含有驱动氯霉素乙酰转移酶（CAT）cDNA的人MxA启动子。通过市售的酶联免疫吸附测定法定量CAT表达。对重组牛IFN-α(1)的反应在0.25至125.0 iu/ml之间呈剂量依赖性，并且显示对I型IFN具有特异性，因为许多其他细胞因子（包括IFN-γ）未观察到显著影响。与用于定量牛样品中IFN活性的传统细胞病变效应降低检测方法相比，这种Mx/CAT报告基因检测在简便性和可靠性方面也具有优势。Mx/CAT报告基因检测成功用于测量从怀孕母牛收集的子宫冲洗液中滋养层来源的I型IFN活性（IFN-τ）。IFN-τ是反刍动物植入前胚胎产生的妊娠识别信号，并且显示在怀孕第12天（0.7±0.14 iu/ml）至第18天（44085.0±14414.2 iu/ml）之间显著增加。相比之下，在人工授精的未怀孕动物中，IFN-τ活性保持在基础水平（0.5 - 0.7 iu/ml）。使用细胞病变效应降低检测方法分析的重复样品与使用Mx/CAT报告基因检测获得的IFN水平相关性良好（P<0.001；r(2)=0.945），证实该报告基因检测可作为标准抗病毒IFN检测的可靠替代方法。
