Nori Human AIF1 ELISA Kit Summary

Alternative names for AIF1: Allograft inflammatory factor 1 (AIF-1), ionized calcium-binding adapter molecule 1 (IBA1)

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: 

Allograft inflammatory factor 1 (AIF-1) also known as ionized calcium-binding adapter molecule 1 (IBA1) is a protein that is encoded by the AIF1 gene.^[1] AIF1 exists in the cytoplasm and is highly evolutionarily conserved. It is also possibly identical to three other proteins, Iba-2, MRF-1 and daintain. However complete functional profiles of all three proteins and how they overlap is unknown.^[2] IBA1 is a 17-kDa EF hand protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells. Iba1 expression is up-regulated in microglia following nerve injury,^[3] central nervous system ischemia, and several other brain diseases. AIF1 was originally discovered in atherosclerotic lesions in a rat model of chronic allograft cardiac rejection. It was upregulated in macrophages and neutrophils in response to IFN-γ.^[4] AIF1 expression has increased in vascular tissue in response to arterial injury, specifically in activated vascular smooth muscle cells in response to IFN-γ, IL-1β, and T-cell conditioned media.^[5] AIF1 enhances growth and promotes proliferation in vascular smooth muscle cells through deregulation of the cell cycle by shortening the cell cycle and altering the expression of cyclins.^[6] Though histologically different, AIF1 promotes the proliferation and activation of endothelial cells (EC).^[7]  AIF1 been shown to interact with kinase p44/42 and PAK1, two previously known signal transduction molecules, in regulating these processes. Allograft Inflammatory Factor 1 is found in activated macrophages that are found in tissues with inflammation. AIF1 levels in healthy humans have been found to positively correlate with metabolic indicators, such as body mass index, triglycerides, and fasting plasma glucose levels. The excess of adipose tissue found in obese patients is found to cause chronic inflammation with an increase in the number of activated macrophages. Subsequently, AIF1 may be an accurate indicator of macrophage activation in the body.^[8] There is also evidence that AIF1 could be a marker for diabetic nephropathy when detected in serum.^[9] It is found in activated macrophages in the pancreatic islets, and has been shown to decrease insulin secretion, while simultaneously impairing glucose elimination.^[10]

References

1. Autieri MV (1996). Biochem Biophys Res Commun. 228 (1): 29–37.
2. Deininger MH, et al. (2002). FEBS Lett. 514 (2–3): 115–21.
3. Ito D, Imai Y, et al. (1998). Brain Res. Mol. Brain Res. 57 (1): 1–9.
4. Utans U, et al. (1995). J. Clin. Invest. 95 (6): 2954–62.
5. Autieri MV, et al. (2000). Arterioscler. Thromb. Vasc. Biol. 20 (7): 1737–44.
6. Autieri MV, Carbone CM (2001). Arterioscler. Thromb. Vasc. Biol. 21 (9): 1421–6.
7. Tian Y, et al. (2009). Am. J. Physiol., Cell Physiol. 296 (2): C256–66.
8. Fukui M, et al. (2012). Metab. Clin. Exp. 61 (7): 1021–5.
9. Fukui M, et al. (2012). Diabetes Res. Clin. Pract. 97 (1): 146–50.
10. Chen ZW, et al. (1997). Proc. Natl. Acad. Sci. U.S.A. 94 (25): 13879–84.