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 bov ine 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, contai ning a human MxA promoter driving a chloramphenicol acetyltransferase (CAT) cDNA. CAT expression was quantified in a commercially available enzyme lin ked immunosorbant assay. The response to recombinant bovine INF-cr, was dos e dependent between 0.25 and 125.0 iu/ml and was shown to be specific for t ype-I IFN as no significant effect was seen with a number of other cytokine s, including IFN-gamma. This Mx/CAT reporter assay also has advantages in t erms of simplicity and reliability over conventional cytopathic effect redu ction assays used to quantify the IFN activity in bovine samples. The Mx/CA T 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-i mplantation embryos and was shown to increase markedly between the 12th (0. 7+/-0.14 iu/ml) and 18th (44 085+/-14 414.2 iu/ml) day of pregnancy, In con trast, IFN-tau activity remained basal (0.5-0.7 iu/ml) in inseminated non-p regnant animals. Duplicate samples analysed using a cytopathic effect reduc tion assay correlated well (P<0.001; r(2)=0.945) with IFN levels obtained u sing the Mx/CAT reporter assay, confirming the reporter assay as a reliable substitute for the standard anti-viral IFN assay. (C) 2001 Elsevier Scienc e B.V. All rights reserved.