Description

Nori Equine BD-3 ELISA Kit Summary

Alternative names for BD-3: beta-Defensin 3, DEFB3

Alternative names for equine: Horse

Q0W9Q0

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: 

Beta defensins are a family of mammalian defensins. The beta defensins are antimicrobial peptides implicated in the resistance of epithelial surfaces to microbial colonization. Defensins are 2-6 kDa, cationic, microbicidal peptides active against many Gram-negative and Gram-positive bacteria, fungi, and enveloped viruses,^[1] containing three pairs of intramolecular disulfide bonds. On the basis of their size and pattern of disulfide bonding, mammalian defensins are classified into alpha, beta and theta categories. Every mammalian species explored thus far has beta-defensins. In cows, as many as 13 beta-defensins exist in neutrophils. However, in other species, beta-defensins are more often produced by epithelial cells lining various organs (e.g. the epidermis, bronchial tree and genitourinary tract). Beta-defensins induce the activation and degranulation of mast cells, resulting in the release of histamine and prostaglandin D2.^[2] β-defensins are coding for genes which impact the function of the innate immune system.^[3] These genes are responsible for production of antimicrobial peptides found in white blood cells such as macrophages, granulocytes and NK-cells, β-defensins are also found in epithelial cells.^[4] β-defensins are cationic and can therefore interact with the membrane of invading microbes, which are negative due to lipopolysaccharides (LPS) and lipoteichoic acid (LTA) found in the cell membrane.^[1] Due to changes in the electric potential, peptides will pass across the membrane and thus aggregate into dimers.^[5] Pore complex will be created as a result of breaking the hydrogen bonds between the amino acids in the terminal end of the strands connecting defensins monomers.^[7] Formation of pore complex will cause membrane depolarization and cell lysis.^[5] Defensins not only have the ability to strengthen the innate immune system but can also enhance the adaptive immune system by chemotaxis of monocytes, T-lymphocytes, dendritic cells and mast cells to the infection site.^[5] Defensins will also improve the capacity of macrophage phagocytosis.^[5]

References

1. White SH, et al. (1995). Opin. Struct. Biol. 5 (4): 521–7.
2. Bensch KW, et al. (1995). FEBS Lett. 368 (2): 331–5.
3. Hellgren O, Sheldon BC (2011). Molecular Ecology Resources 11 (4): 686–692.
4. Ganz T (2003). Rev. Immunol. 3 (9): 710–20.
5. van Dijk A, et al. (2008). Immunol. Immunopathol. 124 (1-2): 1–18.
6. Sugiarto H, Yu PL (2004). Biophys. Res. Commun. 323 (3): 721–7.

Product Citation