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 100 µL of Standard, control, or sample, per well, then add 50 µL of the working solution of Biotin-Conjugate to each well. Cover with the adhesive strip provided and incubate 2 hours at RT. Adequate mixing is very important for good result. Use a mini-vortexer at the lowest frequency.
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 (350 µL) 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 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at RT Avoid placing the plate in direct light.
6. Repeat the aspiration/wash.
7. Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at RT. Avoid placing the plate in direct light.
8. Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
9. 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 Tlymphocytes and natural killer (NK) cells, IFN-γ is now known to be both an inhibitor of viral replication and a regulator of numerous immunological functions. Human 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).  Mouse IFN-γ encodes a 155 amino acid (aa) residue precursor protein with a hydrophobic signal peptide that is cleaved to generate the 133 aa residue mature protein (10).   In solution, IFN-γ has been shown to exist as a non-covalently associated homodimer with topological similarity to IL-10. Mouse IFN-γ shows approximately 40% aa sequence identity to human IFN-γ and there is no cross-reactivity across species (11, 12).  A receptor for 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 IFN-γ(15-18).

Functionally, IFN-γ produces a variety of effects. Produced by CD8+, NK, gd, and TH1 T helper cells, 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 IFN-γ against Toxoplasma and Chlamydia is believed to result from indoleamine 2,3-dioxygenase activity, an enzyme induced by IFN-γ (23). The immunomodulatory effects of IFN-γ are extensive and diverse. In monocyte/macrophages, the activities of 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; downregulation of IL-8 mRNA expression that is upregulated by IL-2; and, with lipopolysaccharide, induction of NO production.  Finally, IFN-γ has been shown to upregulate ICAM-1, but not E-Selectin or VCAM-1, expression on endothelial cells.