Human Proadrenomedullin N-20 terminal peptide (PAMP) ELISA Kit
Cat# AE63301HU
Size : 96Tests
Brand : Abebio
Reagent Preparation
Results demonstration
Assay Procedure Summary
Certificate
Product Details
| Species Reactivity | Human (Homo sapiens) |
| UniProt | N/A |
| Abbreviation | PAMP |
| Alternative Names | ProAM N-terminal 20 peptide; Adrenomedullin; ADM; AM; Contains; RecName; Proadrenomedullin N-20 terminal peptide; ProAM-N20; PAMP; ProAM-N20; ADML_HUMAN. |
| Range | Request Information |
| Sensitivity | Request Information |
| Sample Type | Serum, Plasma, Other biological fluids |
| Detection Method | Sandwich |
| Analysis Method | Quantitive |
| Assay Duration | 1-4.5h |
| Sample Volume | 1-200 μL |
| Detection Wavelengt | 450 nm |
Test principle
This assay employs a two-site sandwich ELISA to quantitate PAMP in samples. An antibody specific for PAMP has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any PAMP present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for PAMP is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of PAMP bound in the initial step. The color development is stopped and the intensity of the color is measured.
Product Overview
Adrenomedullin (ADM) is a peptide associated with pheochromocytoma. Adrenomedullin (AM) is a ubiquitously expressed peptide initially isolated from phaechromyctoma. Adrenomedullin, a hypotensive peptide found in human pheochromocytoma, consists of 52 amino acids, has 1 intramolecular disulfide bond, and shows a slight homology with the calcitonin gene-related peptide. It may function as a hormone in circulation control because it is found in blood in a considerable concentration. The precursor, called preproadrenomedullin, is 185 amino acids long. By RNA-blot analysis, human adrenomedullin mRNA was found to be highly expressed in several tissues. The human AM gene is localized to a single locus on Chromosome 11 with 4 exons and 3 introns. AM and PAMP are potent hypotensive and vasodilatator agents. Numerous actions have been reported most related to the physiologic control of fluid and electrolyte homeostasis. In the kidney, am is diuretic and natriuretic, and both am and pamp inhibit aldosterone secretion by direct adrenal actions. In pituitary gland, both peptides at physiologically relevant doses inhibit basal ACTH secretion. Both peptides appear to act in brain and pituitary gland to facilitate the loss of plasma volume, actions which complement their hypotensive effects in blood vessels.
Components
Reagents | Quantity | Reagents | Quantity |
Assay plate (96 Wells) | 1 | Instruction manual | 1 |
Standard (lyophilized) | 2 | Sample Diluent | 1 x 20 mL |
Biotin-Conjugate (concentrate 100 x) | 1 x 120 μL | Biotin-Conjugate Diluent | 1 x 20 mL |
Streptavidin-HRP (concentrate 100 x) | 1 x 120 μL | Streptavidin-HRP Diluent | 1 x 20 mL |
Wash Buffer (concentrate 25 x) | 1 x 20 mL | Substrate Solution | 1 x 10 mL |
Stop Solution | 1 x 10 mL | Adhesive Films | 4 |
Specificity
This assay has high sensitivity and excellent specificity for detection of Human PAMP. No significant cross-reactivity or interference between Human PAMP and analogues was observed.
Recovery
Matrices listed below were spiked with certain level of recombinant Human PAMP and the recovery rates were calculated by comparing the measured value to the expected amount of Human PAMP in samples.
Sample Type | Number | Recovery range (%) | Average(%) |
Serum | 10 | 90-101 | 96 |
EDTA plasma | 10 | 89-97 | 93 |
Heparin plasma | 10 | 91-99 | 95 |
Precision
Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%
Linearity
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PAMP and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample Type | 1：2 | 1：4 | 1：8 | 1：16 |
Serum | 78-89% | 81-99% | 92-103% | 95-105% |
EDTA plasma | 91-101% | 90-98% | 93-101% | 91-98% |
Heparin plasma | 92-103% | 93-102% | 92-99% | 91-101% |
Stability
The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
Sample collection and storage
Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Kits storage instructions
Store at 2-8°C. Please refer to Instruction Manual.