Human IFN-alpha All Subtype Quantikine ELISA Kit Summary

Product Summary

The Quantikine^® Human IFN-alpha All Subtype Immunoassay is a 4.5 hour solid phase ELISA designed to measure all human IFN-alpha subtypes in cell culture supernates, serum, and plasma. It contains HEK293-expressed recombinant human IFN-alpha 2a and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human IFN-alpha showed linear curves that were parallel to the standard curves obtained using the Quantikine^® kit standards. These results indicate that this kit can be used to determine relative mass values for natural human IFN-alpha subtypes.

Preparation and Storage

Background: IFN-alpha

There are 3 major classes of interferons (IFNs): Type I, Type II and Type III. Interferon alpha (IFN-alpha ), along with IFN-beta, IFN-δ, IFN-epsilon, IFN-kappa, IFN-omega and IFN-tau are all Type I IFNs (1). The sole type II IFN is IFN-gamma. Type III IFNs include IFN-lambda 1, IFN-lambda 2, IFN-lambda 3 and IFN-lambda 4 (2). As a part of the innate immune response, Type I IFNs are rapidly induced in response to viral nucleic acids such as double stranded DNA or RNA (dsDNA, dsRNA) and single stranded RNA (ssRNA), viral glycoproteins, microbial cytosinephosphate-guanosine (CpG) DNA, DNA damage, and chromosomal instability (3,4). 

IFN-alpha subtypes are well described (5-9). There are 15 human IFN-alpha subtypes with 80% amino acid identity (10). The number of IFN-alpha subtypes varies by species with 6 equine subtypes, 17 porcine subtypes, 14 bovine subtypes, and 9 canine subtypes known currently. Human IFN-alpha subtypes include: IFN-alpha 1a, IFN-alpha 1b, IFN-alpha 2a, IFN-alpha 2b, IFN-alpha 4a, IFN-alpha 4b, IFN-alpha 5, IFN-alpha 6 IFN-alpha 7, IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 16, IFN-alpha 17, and IFN-alpha 21. Although there is one known heterodimeric IFN-alpha receptor (IFN-alpha R, described below), each IFN-alpha subtype has been correlated with differing biological activities (7). Variability in biological responses can be attributed to differences in binding affinity and duration, receptor density, feedback responses and intracellular characteristics (11). IFN-alpha responses have been described as robust, especially in the context of viral infection responsiveness by all cells or tunable in a cell type specific manner. 

IFN-alpha signaling is well characterized (2, 5,11,12,13). IFN-alpha is a ligand for IFN alpha R, which includes two subunits IFN alpha R1 and IFN alpha R2. IFN-alpha ligand binding to the ubiquitously expressed IFN alpha R1 triggers a conformational change which allows for the heterodimerization of IFN alpha R1 and IFN alpha R2 (10). Heterodimerization results in the cross phosphorylation of the Janus-activated Kinase 1 (JAK1) on IFN alpha R2 and tyrosine kinase 2 (TYK2) on IFN alpha R1 respectively, as well as the intracellular domain of IFN alpha R1 and IFN alpha 2 (9). The transcription factors Signal transducer and activator of transcription (STAT) 1 and 2 are subsequently recruited to IFN alpha R via their Src homology 2 (SH2) domain and phosphorylated. In the canonical IFN-alpha signaling pathway, phosphoSTAT1/STAT2 heterodimers associate with Interferon Regulatory Factor 9 (IRF9) to form Interferon Stimulated Gene Factor 3 (ISGF3), which translocates into the nucleus to bind to gamma-activated sequences (GASs) or interferon- stimulated response elements (ISREs). This stimulates the transcription of interferonstimulated genes (ISGs). STAT 2 homodimers and monomers can also associate with IRF-9 to form ISGF3-like complex, which also binds to ISRE to stimulate the transcription of ISGs (9). 

Although IFN-alpha is most commonly associated with viral infections, it has been associated with other pathological events. Type 1 interferons represent a standard of care for suppressing Hepatitis B (HBV) or C (HCV) (14). However, it has been associated with neuropsychiatric symptoms such as depression, anhedonia, anxiety and cognitive impairment (15). The role of IFN-alpha in cancer is complex as well. For example, in the context of inflammatory breast cancer, IFN-alpha is upregulated. Although IFN-alpha has been correlated with cellular senescence and apoptosis, some subtypes have been correlated with increased cellular migration and drug resistance.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.