Description

Nori Mouse TLR3 ELISA Kit Summary

Alternative names for TLR3: Toll-like receptor 3 (TLR3), CD283

Q99MB1

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:

Toll-like receptor 3 (TLR3) also known as CD283, is a member of the Toll-like receptor family of pattern recognition receptors of the innate immune system (1). TLRs recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents and mediate the production of cytokines necessary for the development of effective immunity. TLR3 is found in phagosomes (2), where the acidic pH enables binding of internalized double stranded RNA and mRNA from viruses, parasites, and necrotic virally infected cells (3-6, 8, 11). Ligand binding by TLR3 induces receptor dimerization (3, 7, 8) leading to the release of inflammatory cytokines and dendritic cell maturation (4, 6, 9). TLR3 is expressed in dendritic cells, macrophages, microglia, and astrocytes (9-11) and is upregulated by IFNβ and LPS (4, 10). TLR3 expression is also induced by lung fibroblasts and epithelial cells by respiratory syncytial virus infection. TLR3 recognizes double-stranded RNA, a form of genetic information carried by some viruses such as reoviruses. Upon recognition, TLR3 induces the activation of NF-κB to increase production of type I interferons which signal other cells to increase their antiviral defenses. Double-stranded RNA is also recognized by the cytoplasmic receptors RIG-I and MDA-5 (12). TLR3 displays a protective role in mouse models of atherosclerosis. In addition, TLR3 activators show effects on Mouse vascular cells (13).

References

1. Rock FL, et al. Proc. Natl. Acad. Sci. U.S.A. 95 (2): 588–93.
2. Nishiya, T. et al. (2002) J. Biol. Chem. 280:37107.
3. de Bouteiller, O. et al. (2005) J. Biol. Chem. 280:38133.
4. Alexopoulou, L. et al. (2001) Nature 413:732.
5. Aksoy, E. et al. (2005) J. Biol. Chem. 280:277.
6. Kariko, K. et al. (2004) J. Biol. Chem. 279:12542.
7. Choe, J. et al. (2005) Science 309:581.
8. Bell, J.K. et al. (2005) Proc. Natl. Acad. Sci. 102:10976.
9. Farina, C. et al. (2005) J. Neuroimmunol. 159:12.
10. Heinz, S. et al. (2003) J. Biol. Chem. 278:21502.
11. Wang, T. et al. (2004) Nat. Med. 10:1366.
12. Cole JE, et al. Proc Natl Acad Sci U S A 108 (6): 2372–7
13. Choe J, et al. Science 309 (5734): 581–5.

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