Description

Nori Equine ANXA1 ELISA Kit Summary

Alternative names for ANXA1: Annexin A1,

Alternative name for equine: Horse

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: 

Annexin A1 (ANXA1), also known as lipocortin I, is a protein that is encoded by the ANXA1 gene.^[1] ANXA1 belongs to the annexin family of Ca²⁺-dependent phospholipid-binding proteins that are preferentially located on the cytosolic face of the plasma membrane. ANXA1 protein has an apparent relative molecular mass of 40 kDa with phospholipase A2 inhibitory activity. The main mechanism of glucocorticoids’ anti-inflammatory effects is to increase the synthesis and function of ANXA1.^[2] ANXA1 suppresses phospholipase A2, thereby blocking eicosanoid production, and inhibits various leukocyte inflammatory events. In resting conditions, immune cells contain high levels of ANXA1 in their cytoplasm. Following cell activation (for example, by neutrophil adhesion to endothelial-cell monolayers), ANX1 is promptly mobilized to the cell surface and secreted. Annexin A1 promotes neutrophil detachment and apoptosis, and phagocytosis of apoptotic neutrophils by macrophages. On the other hand, it reduces the tendency of neutrophils to penetrate the endothelium of blood vessels. In vitro and in vivo analyses show that exogenous and endogenous ANXA1 counter-regulate the activities of innate immune cells, particularly extravasation and the generation of proinflammatory mediators, which ensures that a sufficient level of activation is reached but not exceeded.^[2] ANXA1 has important opposing properties during innate and adaptive immune responses: it inhibits innate immune cells and promotes T-cell activation. The activation of T cells results in the release of annexin A1 and the expression of its receptor. Higher expression of ANXA1 during pathological conditions could increase the strength of TCR signaling through the mitogen-activated protein kinase signaling pathway, thereby causing a state of hyperactivation of T cells.^[2] Since phospholipase A2 is required for the biosynthesis of the potent mediators of inflammation, prostaglandins, and leukotrienes, ANXA1 may have potential anti-inflammatory activity. Glucocorticoids stimulate production of lipocortin.^[3] In this way, synthesis of eicosanoids are inhibited.

References

1. Wallner BP, et al. (1986). Nature. 320(6057): 77–81.
2. Perretti M, D’Acquisto F (2009). Nat. Rev. Immunol. 9 (1): 62–70.
3. Peers SH, et al. (1993). British Journal of Pharmacology. 108 (1): 66–72.

Product Citation