Hyaluronic acid (HA) ELISA Kit
Katalog-Nummer AE64459GE-480
Size : 5x96tests
Marke : Abebio
Contact local distributor :
Telefonnummer : +1 850 650 7790
Reagent Preparation
Results demonstration
Assay Procedure Summary
Certificate
Product Details
| Species Reactivity | General |
| UniProt | N/A |
| Abbreviation | HA |
| Alternative Names | N/A |
| Range | 6.25-400 ng/mL |
| Sensitivity | 4.17 ng/mL |
| 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 HA in samples. An antibody specific for HA has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any HA present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for HA 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 HA bound in the initial step. The color development is stopped and the intensity of the color is measured.
Product Overview
Hyaluronan (also called hyaluronic acid or hyaluronate) is an anionic, non-sulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. It is unique among glycosaminoglycans in that it is unsulfated, forms in the plasma membrane instead of the Golgi and can be very large with its molecular weight often reaching the millions. One of the chief components of the extracellular matrix, hyaluronan contributes significantly to cell proliferation and migration, and may also be involved in the progression of some malignant tumors. The average 70 kg (154 lbs) person has roughly 15 grams of hyaluronan in their body, one-third of which is turned over (degraded and synthesized) every day. Hyaluronic acid is also a component of the group A streptococcal extracellular capsule, and is believed to play a role in virulence.
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 HA. No significant cross-reactivity or interference between HA and analogues was observed.
Recovery
Matrices listed below were spiked with certain level of recombinant HA and the recovery rates were calculated by comparing the measured value to the expected amount of HA 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 HA 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.