Insulin (INS) BioAssay™ ELISA Kit is a competitive inhibition immunoassay for in vitro quantitative measurement of INS in rat serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.
Principle of the Test:This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for INS has been pre-coated onto the wells of the provided microtiter strips. A competitive inhibition reaction occurs between biotin-labeled INS and unlabeled INS (standards or samples) with the pre-coated antibody specific for INS. After incubation, the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each well and incubated. The amount of bound HRP conjugate is inversely proportional to the concentration of INS in the sample. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of INS in the sample.
Detection Range:123.5-10,000pg/ml
Sensitivity:52.4pg/ml
Intra-Assay CV:<10%
Inter-Assay CV:<12%
Kit Components:*025827A: Microtiter Strips, 1x96 wells, Pre-coated, ready to use*025827B: Standard, 2x1 vial025827C: Standard Diluent, 1x20ml*025827D: Detection Reagent A (green), 1x1vial*025827E: Detection Reagent B (red), 1x120ul025827F: Assay Diluent A, 1x12ml025827G: Assay Diluent B, 1x12ml025827H: TMB Substrate, 1x9ml*025827J: Positive Control 1393.3pg/ml, 1x1vial025827K: Stop Solution, 1x6ml025827L: Wash Buffer, 30X, 1x20ml025827M: Detection Reagent A Diluent, 1x300ul
Precaution:The Stop Solution (025827K) included for use in this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Storage and Stability:Store *025827A, *025827B, *025827D, *025827E and *025827J at -20°C. After reconstitution of *025827J, 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.
Notes on 025827J - Positive Control:Intended Use:The control vials are from a rat serum sample known to contain the target protein. It will provide a positive result in the ELISA assay, which indicates that the kit is optimized and working properly.
Characteristics:The control vials contain rat insulin in rat serum (lyophilized powder). Note: The rat based serum used in this product are from healthy rat of SPF standard (specific pathogen-free animals).
Expected Value:1393.3pg/ml
Usage:Dissolve with 150ul standard diluent and let stand for 10 minutes. Mix gently before use. In the experiment, add 50ul to an appropriate well of the plate and test its concentration along with standards and samples. Note: Upon receipt store Positive Control vial at -20°C. After reconstitution, store Positive Control vial at 4°C and use within 5 days.
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 at 450nm immediately.
