Coagulation Factor XIII (F13) BioAssay™ ELISA Kit (Human) is a sandwich enzyme immunoassay for the in vitro quantitative measurement of F13 in human plasma.
Detection Range:25-1,600pg/ml
Sensitivity:<9.5pg/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 F13. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to F13. 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 F13, 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 F13 in the sample is then determined by comparing the O.D. of the sample to the standard curve.
Storage and Stability:Store *024243A, *024243B, *024243D and *024243E 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:Plasma:Collect plasma using 3.8% sodium citrate (sodium citrate : blood = 1 : 9) 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: Plasma samples require about 50,000-fold dilution. For example, to prepare a 1:50,000 dilution of sample, transfer 20ul of sample to 180ul PBS. This yields a 1:10 dilution. Then, dilute the 1:10 sample by transferring 10ul to 490ul PBS. This yields a 1:500 dilution. Next, dilute the 1:500 sample by transferring 10ul to 990ul PBS. You now have a 1:50,000 dilution of your sample. Mix thoroughly at each stage of the dilution process. Samples should be diluted using 0.01M PBS, pH 7.0-7.2.
Note:1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and contamination
2. Sample hemolysis will influence the results so hemolyzed specimens should not be used
3. When performing the assay, bring samples to RT.
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.
