Description
Description: This assay employs a two-site sandwich ELISA to quantitate IFNE in samples. An antibody specific for IFNE has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IFNE present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for IFNE is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IFNE bound in the initial step. The color development is stopped and the intensity of the color is measured.
Overview: Recently identified interferon-epsilon (IFN-epsilon) belongs to type I interferons. IFN-epsilon is highly and constitutively expressed in the brain, but its biochemical and biological characteristics are poorly understood. In this study, full-length IFN-epsilon cDNA was cloned from human peripheral blood lymphocyte by RT-PCR, and was expressed in Escherichia coli (E coli). Reverse phase high pressure liquid chromatography was used to purify recombinant human IFN-epsilon (rhIFN-epsilon) and to facilitate refolding of the protein. About 0.8 mg of highly purified rhIFN-epsilon, protein was obtained from 100 ml of E. coli culture. Functional study of rhIFN-epsilon demonstrated that the antiviral activity of rhIFN-epsilon was 6 +/- 0.5 x 10(5) IU/mg, which was lower than that of rhIFN-alpha-2b in the WISH-VSV (WISH cells infected with vesicular stomatitis virus) assay system