Quantification of Bioactive Type-I Interferons using a Secreted Embryonic Alkaline Phosphatase Reporter Assay

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.

To measure bioactive type-I interferon using HEK-Blue interferon-alpha/beta reporter cells, first, plate the reporter cells at a density of 50,000 cells per well in a 96-well flat bottom plate to a final volume of 180 microliters. Then, add 20 microliters of the just harvested co-culture supernatant to each well in duplicate. Include a set of internal standard controls, and incubate the plate at 37 degrees Celsius and 5% carbon dioxide for 18 to 22 hours.

To determine the levels of alkaline phosphatase released from the reporter cells, add 180 microliters of QUANTI-Blue solution to each well of a new flat bottom plate. Then, add 20 microliters of the induced HEK-blue interferon-alpha/beta cells supernatants to each well, and incubate the plate at 37 degrees Celsius until color develops in the standard interferon control wells.

The QUANTI-Blue solution changes from pink to purple-blue in the presence of the enzyme. Finally, use a spectrophotometer to evaluate the levels of secreted alkaline phosphatase at 620 to 655 nanometers and determine the concentration of type-I interferon by extrapolation from the linear part of the interferon standard curve.