Afamin (AFM) BioAssay™ ELISA Kit (Human) is a sandwich enzyme immunoassay for in vitro quantitative measurement of AFM in human serum, plasma and other biological fluids.
Detection Range:0.19-12ng/ml
Sensitivity:0.09ng/ml
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 AFM. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to AFM. 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 AFM, 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 AFM in the sample is then determined by comparing the O.D. of the sample to the standard curve.
Kit Components:*023191A: Microtiter Strips, 1x96 wells, Pre-coated, ready to use*023191B: Standard, 2x1vial023191C: Standard Diluent, 1x20ml*023191D: Detection Reagent A (green), 1x120ul*023191E: Detection Reagent B (red), 1x120ul023191F: Assay Diluent A, 1x12ml023191G: Assay Diluent B, 1x12ml023191H: TMB Substrate, 1x9ml023191J: Positive Control, 2.82ng/ml, 1x1vial023191K: Stop Solution, 1x6ml023191L: Wash Buffer, 30X, 1x20ml
Precaution:The Stop Solution (023191K) provided for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Notes on 023191J: Positive Control:Intended Use:The control vial is from a human serum sample known to contain AFM. It will provide a positive result in the ELISA assay, which indicates that the kit is optimized and working properly.
Characteristics:The control vial contains human AFM in human serum (lyophilized powder). Note: The human source material used in this product is screened for HIV, Hepatitis A, B, C, HTLV and Syphilis.
Expected Values:2.82ng/ml
Usage:Dissolve with 150ul standard dilutent and let stand for 10 minutes. If powder is partly insoluble, shake gently until fully dissolved. Mix gently before use. In the experiment, add 100ul to an appropriate well of the plate and test its concentration along with standards and your specimens. Note: Upon receipt store Positive Control vial at -20°C. After reconstitution, store Positive Control vial at 4°C and use within 5 days.
Storage and Stability:Store *023191A, *023191B, *023191D and *023191E and 023191J at -20°C. After reconstitution of *023191J, store at 4°C and use within 5 days. 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 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.
