Materials Provided:

• Pre-coated microtiter plate
• Plate Sealers
• Wash Solution Concentrate
• Mouse IFN-Alpha 4 Standard, 10,000 pg/ml
• Sample Diluent
• Antibody Concentrate
• Antibody Diluent
• HRP Conjugate Concentrate
• HRP Diluent
• TMB Substrate Solution
• Stop Solution

Additional Materials Required (Not Provided) 

• Microplate reader capable of reading an OD at a wavelength of 450 nm
• Variable volume microtiter pipettes
• Adjustable multichannel 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)
• Plate shaker

 

Representative Mouse IFN-Alpha Standard Curve in Sample Diluent

 

 

Intra and Inter-Assay CVs to measure Precision
Concentration (pg/ml)	Endogenous Level
	Low	Medium	High
Intra-Assay CV (%)	4.4	3.3	2.8
Inter-Assay CV (%)	7.8	5.8	8.7

 

 

Spike Recovery
Dilution	Average Concentration (pg/ml)		% Linear Adjusted Dilution
	Not Adjusted for Dilution	Adjusted for Dilution
2	39.5	79.1	-
4	19.1	76.6	96.8
8	9.7	77.3	97.8
16	4.9	78.8	99.7
32	2.5	80.7	102.0
64	1.2	77.7	98.2
Avg	-	78.4	98.9

 

 

Endogenous Mouse IFN-Alpha Quantified in Plasma or Serum From Three Mouse Strains Using PBL’s High Sensitivity Mouse IFN-Alpha All Subtype ELISA

Background

Interferons (IFNs) are an extensive set of cytokines exhibiting pleiotropic activities and playing major roles in both innate and adaptive immunity. The Type I IFNs genes consist of multiple IFN-Alpha genes, at least one IFN-Beta gene in most vertebrates, and a few other family members including limitin in the mouse and IFN-Omega and -Epsilon in mice and humans. In the mouse, fourteen (14) IFN-Alpha genes have been cloned which exhibit at least 75% identity at the protein level. In the therapeutic setting, IFN-Alpha is used to treat viral diseases and certain cancers and is therefore frequently studied in mouse and other animal models of these diseases. Several lines of evidence indicate that IFN-Alpha proteins may be involved in the development of specific autoimmune diseases and that treatment with IFN-Alpha blocking antibodies may comprise a useful therapeutic strategy.

The expression of interferon genes (and therefore, proteins) is regulated by a group of interferon regulatory factors, i.e., the IRF family of proteins. Cellular levels of IRF3 and IRF7 regulate the expression of individual IFN-Alpha subtypes with most IFN-Alpha subtype genes requiring IRF7 for expression. IRF7 protein is either constitutively expressed or inducible, depending on cell type. In certain mouse cell lines, IRF7 expression is induced in particular by at least IFN-Beta and IFN-Alpha4 which then leads to expression of the other IFN-Alpha subtype genes. Additionally, murine IFN-Beta may be requisite for fibroblast synthesis of IFN-α but may not be required for other cells’ synthesis of IFN-Alpha. The IRF3/IRF7 signaling cascade is a focal point for initial and progressive cellular responses to pathogens where hundreds of genes are subsequently regulated in a temporally coordinated cascade.

Plasmacytoid dendritic cells (PDCs) are highly efficient producers of IFN-Alpha, but a wide variety of cells can also produce lower levels of these protein subtypes. The particular IFN-Alpha subtype genes that are expressed appear to be somewhat cell and stimulus-specific and, based on the results published from several gene induction studies, the time courses of induction of the individual protein subtypes are expected to differ markedly.

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