Rat IFN-gamma ELISA Assay

$430.00

The Eagle Biosciences Rat Interferon Gamma (IFN-γ) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of rat γ-interferon (IFN-γ) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Rat Interferon Gamma (IFN-γ) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Rat IFN-gamma ELISA Assay

The Rat IFN-gamma ELISA Assay is For Research Use Only

Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 31.25 – 2000 pg/mL
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: IFN-g, Interferon Gamma, IFN-γ, Interferon IFN-γ


SAMPLE COLLECTION AND STORAGE
1. Cell Culture Supernates – Remove particulates by centrifugation.
2. Serum – Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum, avoid hemolysis and high blood lipid samples.
3. Plasma – Recommended EDTA as an anticoagulant in plasma. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection.
4. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.
5. Dilute samples at the appropriate multiple (recommended to do pre-test to determine the dilution factor).
Note: Normal rat serum or plasma samples are suggested to make a 1:2 dilution.


Assay Principle

The Rat Interferon Gamma (IFN-g) ELISA kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for interferon gamma (IFN-γ) has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any interferon gamma (IFN-γ) present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for interferon gamma (IFN-γ) is added to the wells and binds to the combination of capture antibody-IFN-γ in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of interferon gamma (IFN-γ) present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven interferon gamma (IFN-γ) standard dilutions and interferon gamma (IFN-γ) sample concentration determined.


Related Products

Rat TNF-Alpha ELISA Assay Kit
Interferon Gamma ELISA Assay
Mouse Interferon Gamma ELISA Assay

Additional Information

Assay Procedure

  1. Prepare all reagents and working standards as directed in the previous sections.
  2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
  3. Add 100mL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
  4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350mL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
  5. Add 100 mL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hours at 37°C.
  6. Repeat the aspiration/wash.
  7. Add 100 mL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
  8. Repeat the aspiration/wash.
  9. Add 100 mL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
  10. Add 100 mL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  11. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length; 610-650nm is acceptable)

Assay Background


Interferon gamma (IFN-γ) is a multifunctional protein first observed as an antiviral activity in cultures of Sindbis virus-infected human leukocytes stimulated by PHA (1). Produced by T-lymphocytes and natural killer (NK) cells, interferon gamma (IFN-γ) is now known to be both an inhibitor of viral replication and a regulator of numerous immunological functions. Human interferon gamma (IFN-γ) is reported to be active only on human and non-human primate cells (5). The biochemistry and biological activities of the interferons have been extensively reviewed (2-9).

Rat interferon gamma (IFN-γ) cDNA encodes a 156 amino acid (aa) residue precursor protein with a putative 19 aa residue signal peptide that is cleaved to generate the mature protein which contains two potential N-glycosylation sites (10-12). Rat IFN-γ shares approximately 87% and 39% amino acid sequence identity with mouse interferon gamma (IFN-γ) and human interferon gamma (IFN-γ), respectively. Consistent with their degrees of shared homology, rat
A receptor for interferon gamma (IFN-γ) has been identified and its gene localized to chromosome 6 (13,14). Apparently the product of a single gene, the receptor is a single chain 90 kDa glycoprotein that shows a high degree of species-specific binding of interferon gamma (IFN-γ) (15-18).

Functionally, interferon gamma (IFN-γ) produces a variety of effects. Produced by CD8+, NK, gd, and TH1 T helper cells, interferon gamma (IFN-γ) has documented antiviral, antiprotozoal and immunomodulatory effects on cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes (19-22) he antiprotozoal activity of interferon gamma (IFN-γ) against Toxoplasma and Chlamydia is believed to result from indoleamine 2,3-dioxygenase activity, an enzyme induced by interferon gamma (IFN-γ) (23).The immunomodulatory effects of interferon gamma (IFN-γ) are extensive and diverse. In monocyte/macrophages, the activities of interferon gamma (IFN-γ) include: increasing the expression of class I and II MHC antigens; increasing the production of IL-1, platelet-activating factor, H2O2, and pterin; protection of monocytes against LAK cell-mediated lysis; down regulation of IL-8 mRNA expression that is up regulated by IL-2; and, with lipopolysaccharide, induction of NO production.  Finally, interferon gamma (IFN-γ) has been shown to upregulate ICAM-1, but not E-Selectin or VCAM-1, expression on endothelial cells.

Manual

Product Manual


Publications

References


1.    Wheelock, E.F. (1965) Science 149:310.
2.    Ijzermans, J.M. and R.L. Marquet (1989) Immunobiol. 179:456.
3.    Mogensen, S.C. and J.L. Virelizier (1987) Interferon 8:55.
4.    Grossberg, S.E. et al. (1989) Experientia 45:508.
5.    Adolf, G.R. (1985) Oncology (Suppl. 1) 42:33.
6.    Samuel, C.E. (1991) Virology 183:1.
7.    Pellegrini, S. and C. Schindler (1993) Trends Biochem. Sci. 18:338.
8.    Reiter, Z. (1993) J. Interferon Res. 13:247.
9.    Boehm, U. et al. (1997) Annu. Rev. Immunol. 15:749.
10.    Puddu, P. et al. (1997) J. Immunol. 159:3490.
11.    Yoshimoto, T. et al. (1997) Proc. Natl. Acad. Sci. USA 94:3948.
12.    Dijkema, R. et al. (1986) Meth. Enzymol. 119:453.
13.    Rashidbaigi, A. et al. (1986) Proc. Natl. Acad. Sci. USA 83:384.
14.    Pfizenmaier, K. et al. (1988) J. Immunol. 141:856.
15.    Aguet, M. et al. (1988) Cell 55:273.
16.    Fischer, D.G. et al. (1988) J. Biol. Chem. 263:2632.
17.    Calderon, J. et al. (1988) Proc. Natl. Acad. Sci. USA 85:4837.
18.    Paliard, X. et al. (1988) J. Immunol. 141:849.
19.    Christmas, S.E. (1992) Chem. Immunol. 53:32.
20.    Locksley, R.M. and P. Scott (1991) Immunoparasitology Today A58-A61.
21.    Billiau, A. and R. Dijkmans (1990) Biochem. Pharmacol. 40:1433.
22.    Sen, G.C. and P. Lengyel (1992) J. Biol. Chem. 267:5017.
23.    Gusella, G.L. et al. (1993) J. Immunol. 151:2725.

Citations


Morsy, L. et. al. “Attenuation of renal ischemia/reperfusion injury by açaí extract preconditioning in a rat model.” Life Sciences 2014, 123:35–42.