Typical Standard Curves of Human IFN-Alpha (see Technical Data Sheet)

Linearity of Dilution in Three Different Culture Medias (see Technical Data Sheet)

Parallelism of Endogenous Samples (see Technical Data Sheet)

sIFNAR2 Receptor Interference (see Technical Data Sheet)

Endogenous Levels of IFN-Alpha Quantified in Sendai Virus infected A549 Cells (see Technical Data Sheet for more detail)

Endogenous Levels of IFN-Alpha Quantified in Sendai Virus infected U937 (see Technical Data Sheet)

Typical Standard Curves of All 12 Human IFN-Alpha Subtypes (see Protocol)

LLOQ and LLOD of All 12 Human IFN-Alpha Subtypes using PBL's High Sensitivity Human IFN Alpha All Subtype TCM ELISA (41135)

Human IFN-Alpha All Subtype ELISA Kit, High Sensitivity (TCM)

Catalog Number: 41135

This ELISA quantitates the amount of IFN-Alpha in tissue culture media (TCM) with an LLOQ of 1.95 pg/ml. This kit provides sensitive and total detection of all human IFN-Alpha subtypes.

Product Name: VeriKine-HS Human Interferon-Alpha All-Subtype ELISA Kit (TCM)

$545.00

$645.00

Matrix Compatibility	Tissue Culture Media (TCM)
Assay Range	1.95 - 125 pg/ml
LLOQ	1.95 pg/ml Need more sensitivity? Check out our Sample Testing Services
Assay Length	20 hours 30 minutes - 24 hours 30 minutes
Specificity	Human IFN-α

The VeriKine-HS Human Interferon Alpha All Subtype TCM ELISA Kit is specifically formulated to detect all human IFN-Alpha subtypes. This kit quantitates all human IFN alpha subtypes in tissue culture media (TCM) using a sandwich immunoassay format. The kit is based on an ELISA with biotinylated-detection antibody and streptavidin-conjugated horseradish peroxidase (HRP). Tetramethyl-benzidine (TMB) is the substrate. The assay is based on the international reference standard for human interferon alpha provided by the National Institutes of Health.

*For complex biological matrices such as plasma, healthy sera, and autoimmune disease sera, we recommend our Human IFN-Alpha All Subtype ELISA Kit, High Sensitivity (Cat. No. 41115-1).

CVs and Spike Recovery	Inter-Assay: ≤ 10% Intra-Assay: ≤ 8% Average Spike Recovery: 105%
Cross-reactivity	Cross-reacts with Cynomolgus/Rhesus IFN-α (13%) No cross-reactivity against Human IFN-β, IFN-γ, or IFN-ω Mouse or Rat IFN-α, IFN-β or IFN-γ Pig IFN-α Bovine IFN-τ
Storage	2-8°C
Expiration Date	One year from the date of manufacture
Shipping Condition	Wet Ice

Materials Provided

Pre-coated microtiter plate
Plate Sealers
Wash Solution Concentrate
Human Interferon Alpha Standard, 10,000 pg/ml
Assay Buffer
Standard Diluent
Antibody Diluent
Antibody Concentrate
HRP Conjugate Concentrate
HRP Diluent
TMB Substrate Solution
Stop Solution

Additional Materials Required (Not Provided)

Microtiter plate reader capable of reading a wavelength of 450 nm
Plate shaker (recommended shaking capability of 550 rpm)
Variable volume microtiter pipettes
Adjustable multi-channel pipette (50-300 μl)
Reagent reservoirs
Wash bottle or plate washing system
Distilled or deionized water
Serological pipettes (1, 5, 10 or 25 ml)
Disposable pipette tips (polypropylene)

Tips, Tools and Troubleshooting:

Human IFN-Alpha ELISA Subtype Specificities Comparison Chart

Subtype	LLOQ
Representative Human IFN-Alpha All Subtype Detection
α1 (αD)	2.06
α2a (αA)	1.41
α4a (αM1)	2.03
α5 (αG)	1.20
α6 (αK)	0.82
α7 (αJ1)	1.93
α8 (αB2)	2.67
α10 (αC)	1.98
α14 (αH)	3.06
α16 (αWA)	2.39
α17 (αI)	2.26
α21 (αF)	4.96

Representative Standard Curves of Human IFN-Alpha Subtypes

Intra-Assay Precision
TC Media Spike Sample	1	2	3
n	20	20	20
Mean (pg/ml)	2.39	12.90	87.16
Standard Deviation	0.13	0.46	4.58
CV (%)	5.4	3.6	5.3

Inter-Assay Precision
TC Media Spike Sample	1	2	3
n	9	9	9
Mean (pg/ml)	3.29	15.13	93.69
Standard Deviation	0.16	0.83	6.07
CV (%)	4.9	5.5	6.5

Intermediate Precision
TC Media Spike Sample	1	2	3
n	9	9	9
Mean (pg/ml)	3.29	15.30	94.00
Standard Deviation	0.44	1.75	7.37
CV (%)	13.4	11.4	7.8

Spike Recovery
TC Media Spike Sample	1	2	3
n	25	25	25
Average Recovery (%)	109	102	104
Range	86-143	81-122	90-120

Linearity

Human IFN-Alpha was spiked into three tissue culture media (RPMI, DMEM and MEM) at 80 pg/ml and then serially diluted with Standard Diluent to assess assay linearity.

Parallelism of Endogenous Samples

Five endogenous samples were tested on Cat No. 41135 and assayed in duplicate. Samples were serially diluted in TCM to the 32nd dilution, which still lies within standard curve range. Percent recoveries are 100±20% of the neat value. Cat. No. 41135 has acceptable parallelism. Error bars indicate standard deviation.

sIFNAR2 Receptor Interference

To test whether the soluble IFN Alpha/Beta Receptor 2 (sIFNAR2) receptor interferes with the detection of IFN-Alpha in Cat No. 41135, two concentrations of standard (1.6 ng/ml and 16 ng/ml) were pre-incubated with sIFNAR2 for 1 hour at room temperature, prior to proceeding with the assay. Concentrations were interpolated from assay calibrator. Standard curves preincubated with sIFNAR2 were backfitted against standard curves without sIFNAR2. Error bars indicate standard deviation. (n=2)

Endogenous Levels of IFN-Alpha Quantified in Sendai Virus (A549)

A549 cells were infected with 15 HA units of Sendai Virus (SeV). Supernatants were collected at five time points post infection (2, 6, 24, 48, and 72 hours). Samples were assayed in Cat. No. 41135, Competitor A’s, and Competitor B’s plates; there was no quantification on Competitor B’s plate. 24, 48 and 72 hour time points were prediluted 1:10 in DMEM prior to start of assay for 41135 only. The standard curve and samples were assayed in duplicate. Sample concentrations were interpolated from assay calibrators from each respective vendor. Error bars indicate standard deviation.

Endogenous Levels of IFN-Alpha Quantified in Sendai Virus (U937)

U937 cells were infected with 1.5 HA units of Sendai Virus (SeV). Supernatants were collected at four timepoints post infection (6, 24, 48, and 72 hours). Samples were prediluted to 1:50 final dilution in RPMI prior to start of assay, except for 6 hour timepoint. The standard curve and samples were assayed in duplicate. Sample concentrations were interpolated from assay calibrators from each respective vendor. Error bars indicate standard deviation.

Background

Interferons (IFNs) are a family of mammalian cytokines initially characterized by their ability to inhibit viral infection. They are synthesized and secreted by most cell types in response to pathogens. In addition to their antiviral properties, IFNs have also been shown to exhibit anti-proliferative, immunomodulatory, and many other activities.

In humans, IFN-α refers to a family of proteins comprised of 12 highly homologous protein subtypes (greater than 85% by amino acid sequence), encoded by 13 genes, that exhibit pleiotropic biologic activities. Measuring the levels of the 12 different human IFN-α subtypes is essential for understanding their triggers and subsequent effects on the immune system. The ability to measure all the subtypes may provide an indication of the total IFN-α level.

3 Citations:

Sugimoto, A. et al., (2023), "Identification of novel lactic acid bacteria with enhanced protective effects against influenza virus", PLoS One, 18(8):e0273604, PMID: 37556447, DOI: 10.1371/journal.pone.0273604 (link)

Killarney, S. et al., (2023), "Executioner caspases restrict mitochondrial RNA-driven Type I IFN induction during chemotherapy-induced apoptosis", Nat. Commun. 14(1):1399, PMID: 36918588, DOI: 10.1038/s41467-023-37146-z (link)

Dowling, D.J. et al., (2022), "development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination", Sci. Rep. 12(1):16860, PMID: 36258023, DOI: 10.1038/s41598-022-20346-w (link)

Background Literature:

Staehelin et al. (1981) "A Rapid Quantitative Assay of High Sensitivity for Human Leukocyte Interferon with Monoclonal Antibodies" in Methods in Enzymology, Vol. 79 (Pestka, ed.), Academic Press, New York, 589-595.

Kelder et al. (1986) "A Sandwich Radioimmunoassay for Human IFNa" in Methods in Enzymology, Vol. 119 (Pestka, ed.), Academic Press, New York, 582-587.

Human IFN-α international reference standard provided by the NIH, reference no. Gxa01-901-535. Pestka (1986) "Interferon Standards and General Abbreviations" in Methods in Enzymology, Vol. 119 (Pestka, ed.), Academic Press, New York, 14-23.

Rubinstein et al. (1981) "Human Leukocyte Interferon: Isolation and Characterization of Several Molecular Forms," Arch. Biochem. Biophys. 210, 307-318.

Hobbs et al. (1982) "Purification and Characterization of Interferons from a Continuous Myeloblastic Cell Line," J. Biol. Chem. 257, 4071-4076.

Documentation

Certificate of Analysis (CoA), Protocol, Safety Data Sheet (SDS), and Technical Data Sheet (TDS)

41135 CoA & Protocol

41135-1 Certificate of Analysis & Protocol

41135 TDS

41135 Technical Data Sheet

41135 SDS

41135 Safety Data Sheet

41135 Protocol

41135 Protocol (Full)

Human IFN-Alpha All Subtype ELISA Kit, High Sensitivity (TCM)