Abstract：
: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.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Fray MD,Mann GE,Charleston Bdoi
10.1016/s0022-1759(00)00359-8keywords:
subject
Has Abstractpub_date
2001-03-01 00:00:00pages
235-44issue
1-2eissn
0022-1759issn
1872-7905pii
S0022175900003598journal_volume
249pub_type
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