Validation of an Mx/CAT reporter gene assay for the quantification of bovine type-I interferon

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-α 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-γ. 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-τ) in uterine flushings collected from pregnant cows. IFN-τ 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 (44 085.0±14 414.2 iu/ml) day of pregnancy. In contrast, IFN-τ 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