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-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.
Fray
M D
MD
Institute for Animal Health, Compton, Berkshire, RG20 7NN, Newbury, UK. martin.fray@bbsrc.ac.uk
Mann
G E
GE
Charleston
B
B
D000818:Animals; D001681:Biological Assay; D002417:Cattle; D015500:Chloramphenicol O-Acetyltransferase; D005260:Female; D017930:Genes, Reporter; D006801:Humans; D007370:Interferon Type I / Q000032:analysis* / Q000235:genetics; D011247:Pregnancy; D011994:Recombinant Proteins
D007370:Interferon Type I; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase