The Rat Kisspeptin 1 (KISS1) ELISA Kit is a competitive inhibition immunoassay for the in vitro quantitative measurement of KISS1 in rat serum, plasma and other biological fluids.
Test Principle:This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for KISS1 has been pre-coated onto a microplate. A competitive inhibition reaction occurs between biotin-labeled KISS1 and unlabeled KISS1 (standards or samples) for limited binding sites on the pre-coated antibody specific for KISS1. 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 inversely proportional to the concentration of KISS1 in the sample. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of KISS1 in the sample.
Storage and Stability:Store *026390A, *026390B, *026390D and *026390E 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: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.
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.
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 room temperature.
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
