Quantification of Bioactive Type-I Interferons using a Secreted Embryonic Alkaline Phosphatase Reporter Assay
Trascrizione
To quantify the bioactive type-I interferons, such as interferon-alpha and interferon-beta, in a test sample, take a multi-well plate containing engineered reporter cells. These cells express the secreted embryonic alkaline phosphatase, SEAP, reporter gene under the control of the interferon-alpha and beta inducible promoter.
Add the test sample containing an unknown type-I interferon concentration. Pipette a range of human type-I interferon concentrations to other wells, which act as standard controls. Incubate.
Type-I interferon in the test well binds to a specific cell surface receptor, activating the Janus kinases. These protein kinases phosphorylate signal transducers and activators of transcription, STAT, proteins, which dimerize and form a complex with a specific interferon regulatory factor.
Upon entering the nucleus, the complex binds to a specific DNA sequence in the interferon-alpha and beta inducible promoter, activating SEAP gene transcription and producing the SEAP enzymes, which are secreted into the media.
Transfer the SEAP-containing media to multi-well plate wells having a SEAP-specific substrate. SEAP hydrolyzes the pink substrate, producing a purple-blue-colored product.
Using a microplate reader, measure the product's absorbance in the test and standard wells, indicative of SEAP levels and Type-I interferon concentration.
From the plotted standard type-I interferon curve, determine the bioactive type-I interferon concentration in the test sample.
