Description

Nori Rabbit IFNa ELISA Kit Summary

Alternative names for IFNa: Interferon alpha,

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Interferons (IFNs) are proteins made and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites or tumor cells, and are very important for fighting viral infections. They allow for communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors. IFNs belong to the large class of glycoproteins known as cytokines and are named after their ability to “interfere” with viral replication within host cells. IFNs have other functions: activation of  immune cells, such as natural killer cells and macrophages; increasing recognition of infection or tumor cells by up-regulating antigen presentation to T lymphocytes; and increasing the ability of uninfected host cells to resist new infection by virus. Certain symptoms, such as aching muscles and fever, are related to the production of IFNs during infection. About ten distinct IFNs have been identified in mammals and are typically divided among three IFN classes: Type I, Type II and Type III IFN and IFNα belongs to type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.^[1]  In addition to the JAK-STAT pathway, IFNs can activate several other signaling cascades. Both type I and type II IFNs activate a member of the CRK family of adaptor proteins called CRKL, a nuclear adaptor for STAT5 that also regulates signaling through the C3G/Rap1 pathway.^[2] Type I IFNs further activate p38 mitogen-activated protein kinase (MAP kinase) to induce gene transcription. ^[2]  The immune effects of interferons have been exploited to treat several diseases. Agents that activate the immune system, such as small imidazoquinoline molecules that activate TLR7, can induce IFN-α.  Patients with recurrent melanomas receive recombinant IFN-α2b.^[3] IFN-α 2b is also being used for treatment of ocular surface squamous neoplasia (OSSN). Both hepatitis B and hepatitis C are treated with IFN-α, often in combination with other antiviral drugs.^[4]

References

1. de Weerd NA, et al. (2007). J Biol Chem 282 (28): 20053–20057.
2. Platanias, L. C. (May 2005). Nature reviews. Immunology 5 (5): 375–386.
3. Hauschild, A. et al. (2008). Cancer 112 (5): 982–994.
4. Cooksley, W. G. (Mar 2004). MedGenMed : Medscape general medicine 6 (1): 16.

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