Nori Mouse alpha-Defensin 1 ELISA Kit Summary

Alternative names for alpha-defensin 1: DEFA1, Defensin, alpha 1, alpha defensin 1, neutrophil peptide 1 (HNP-1), neutrophil defensin 1

P11477

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:

Defensin, alpha 1 also known as alpha defensin 1, neutrophil peptide 1 (HNP-1) or neutrophil defensin 1 is a protein that is encoded by the DEFA1 gene.^[1] Human alpha defensin 1 belongs to the alpha defensin family of antimicrobial peptides. Defensins are a family of microbicidal and cytotoxic peptides thought to be involved in host defense. They are abundant in the granules of neutrophils and also found in the epithelia of mucosal surfaces. Members of the defensin family are highly similar in protein sequence and distinguished by a conserved cysteine motif. Defensin, alpha 1 is found in the microbicidal granules of neutrophils and likely plays a role in phagocyte-mediated host defense. It differs from the defensins, alpha 2 and alpha 3 by only one amino acid. HNPs are generated as 94 amino acids preproHNPs, which are co-translationally cleaved to 75 amino acids pro-peptides with a N-terminal prosegment having a negative charge that neutralizes the highly positively charged C terminal peptide. Processing of proHNPs occurs mainly in late promyelocytes, where the 75 amino acids proHNPs are cleaved to a 56 amino acids intermediate form and onward to 29-30 amino acids mature peptides designated HNPs.^[2] Cationic 29-30 amino acids HNPs associate with the negatively charged proteoglycan serglycin and translocate to azurophil granules.^[3] At later stages of granulocytic differentiation in which HNP expression peaks (i.e. myelocytes and metamyelocytes), proHNPs are not cleaved, rendering the peptides overall neutral. This prevents binding to serglycin and most proHNP is accordingly secreted into the bone marrow plasma although some is retained in specific granules.^[4]

References

1. Aldred PM, et al. (2005). Hum Mol Genet. 14 (14): 2045–52.
2. Valore EV, Ganz T (1992). Blood. 79 (6): 1538–44.
3. Glenthøj A, et al. (2011). Blood. 118 (16): 4440–8.
4. Faurschou M, et al. (2005). Journal of leukocyte biology. 78 (3): 785–93.