Aggrecan (AGC) BioAssay™ ELISA Kit (Human)

Blue Ice

38220000

The Human Aggrecan (AGC) ELISA kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of AGC in human serum, plasma and other biological fluids.

Detection Range:
6.25-100ng/ml

Sensitivity:
<2.27ng/ml

Precision:
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Test Principle:
The microtiter plate provided in this kit has been pre-coated with an antibody specific to AGC. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to AGC. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain AGC, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of AGC in the sample is then determined by comparing the O.D. of the sample to the standard curve.

Kit Components:
*023193A: Microtiter Plate, 1x96 wells, Pre-coated; ready to use.
*023193B: Standard, 2x1vial
023193C: Standard Diluent, 1x20ml
*023193D: Detection Reagent A, 1x120ul
*023193E: Detection Reagent B, 1x120ul
023193F: Assay Diluent A, 1x12ml
023193G: Assay Diluent B, 1x12ml
023193H: TMB Substrate, 1x9ml
023193K: Stop Solution, 1x6ml
023193L: Wash Buffer, 30X, 1x20ml

Storage and Stability:
Store *023193A, *023193B, *023193D and *023193E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. 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.

Materials Required But Not Supplied:
1. Microplate reader with 450 ± 10nm filter.
2. Precision single or multi-channel pipettes and disposable tips.
3. Eppendorf Tubes for diluting samples.
4. Deionized or distilled water.
5. Absorbent paper for blotting the microtiter plate.
6. Container for Wash Solution

Sample Preparation and Storage:
Serum:
Use a serum separator and allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Plasma:
Collect plasma using EDTA or heparin as anticoagulant. Centrifuge samples for 15 minutes at 1000xg at 2-8°C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.

Note: Serum/plasma requires about 100-fold dilution (example: 10ul sample + 990ul PBS). Samples should be diluted using 0.01M PBS, pH 7.0-7.2.

Other Biological Fluids:
Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles.

Assay Procedure Summary:
1. Prepare all reagents, samples and standards.
2. Add 100ul standard or sample to each well. Incubate 2 hours at 37°C.
3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C.
4. Aspirate and wash 3 times.
5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C.
6. Aspirate and wash 5 times.
7. Add 90ul TMB Substrate. Incubate 15-25 minutes at 37°C.
8. Add 50ul Stop Solution. Read at 450nm immediately.