This Hyaluronic Acid (HA) BioAssay™ ELISA Kit (General) uses a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of HA in serum, plasma and other biological fluids.
Range:4.94-400ng/ml
Sensitivity:<1.81ng/ml
Precision:Intra-Assay: CV<10%Inter-Assay: CV<12%
Specificity:This assay has high sensitivity and excellent specificity for detection of Hyaluronic Acid (HA). No significant cross-reactivity or interference between Hyaluronic Acid (HA) and analogues was observed.
Test Principle:This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to hyaluronic acid has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled hyaluronic acid and unlabeled hyaluronic acid (standards or samples) with the pre-coated antibody specific to hyaluronic acid. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of hyaluronic acid in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of hyaluronic acid in the sample.
Kit Components:Microtiter Plate, 1x96 wells. Pre-coated; ready to use
Standard, 2x1vial Standard Diluent, 1x20mlDetection Reagent A, 1x vialDetection Reagent B, 1x120ul Assay Diluent A, 1x12mlAssay Diluent B, 1x12mlTMB Substrate, 1x9mlStop Solution: 1x6mlReagent Diluent, 1x300ulWash Buffer, 30X, 1x20ml
Storage and Stability:Store Microtiter Plate, Standard, and Detection Reagents A and B at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months after receipt. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.
Assay Procedure Summary:1. Prepare all reagents, samples and standards
2. Add 50ul standard or sample to each well, then add 50ul prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37°C
3. Aspirate and wash 3 times
4. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C
5. Aspirate and wash 5 times
6. Add 90ul Substrate Solution. Incubate 15-25 minutes at 37°C
7. Add 50ul Stop Solution. Read absorbance at 450nm immediately.
