Description

Nori Canine IFNg ELISA Kit Summary

Alternative names for IFNg: Interferon gamma,

Alternative name for canine: dog

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: 

IFN-γ is a dimerized soluble cytokine that is the only member of the type II class of interferons (1). It plays key roles in both the innate and adaptive immune response against viral and intracellular bacterial infections and for tumor control (2). It alters transcription in up to 30 genes producing a variety of physiological and cellular responses. IFN-γ activates the cytotoxic activity of innate immune cells such as macrophages and NK cells (3,4). IFN-γ production by NK cells and antigen-presenting cells (APCs) promotes the cell mediated adaptive immunity by inducing IFN-γ production by T lymphocytes, increased class I and class II MHC expression, and enhancing peptide antigen presentation (3). The anti-viral activity of IFN-γ is due to its induction of PKR and other regulatory proteins. Binding of IFN-γ to the IFNGR1/IFNGR2 complex promotes dimerization of the receptor complexes to form (IFNGR1/IFNGR2)₂ -IFN-γ dimer. Binding induces a conformational change in receptor intracellular domains and signaling involves Jak1, Jak2 and Stat1 (5). The critical role of IFN-γ in amplification of immune surveillance and function is supported by increased susceptibility to pathogen infection by IFN-γ or IFNGR knockout mice and in humans with inactivating mutations in IFNGR1 or IFNGR2. IFN-γ also appears to have a role in atherosclerosis (6).

References

1. Gray PW and Goeddel DV (1982). Nature 298 (5877): 859–63.
2. Schroder K, et al (2004). J. Leukoc. Biol. 75 (2): 163–89.
3. Schroder, K. et al. (2004) J Leukoc Biol 75, 163-89.
4. Martinez, F.O. et al. (2009) Annu Rev Immunol 27, 451-83.
5. Kotenko, S.V. et al. (1995) J Biol Chem 270, 20915-21.
6. McLaren, J.E. and Ramji, D.P. (2009) Cytokine Growth Factor Rev 20, 125-35.

Product Citations