Urocortin 1 ELISA 试剂盒

1. Preparation of Calibrator Solutions: Reconstitute the Urocortin 1 Calibrator with 1 mL of Buffer Solution, giving a 100 ng/mL Calibrator Solution after reconstitution. 0.1 mL of the reconstituted Calibrator Solution is diluted with 0.1 mL of Buffer Solution to yield a 50 ng/mL Calibrator Solution. Repeat the serial dilution to make Calibrator Solutions at 25, 12.5, 6.25, 3.125, and 1.563 ng/mL. Buffer Solution is used as the zero calibrator (0 ng/mL).
   2. Preparation of Labeled Antigen: Reconstitute Labeled Antigen with 6 mL of distilled water.
   3. Preparation of Wash Solution: Dilute 50 mL of Wash Solution Concentrate to 1,000 mL with distilled or de-ionized water.
   4. Other reagents are ready for use.

1. Warm the reagents and samples to room temperature (20
   30°C) before beginning the test.
   2. Add 0.3 mL of diluted Wash Solution into the wells and aspirate the Washing Solution in the wells. Repeat this washing procedure twice, for a total of 3 wash steps. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
   3. Add 40 µL Buffer Solution into the wells, then add 10 µL of the prepared Calibrator Solutions (0, 1.563, 3.125, 6.25, 12.5, 25, 50, and 100 ng/mL) or samples into wells. Add 50 µL of Labeled Antigen into the wells. The total pipetting time of calibrator solutions and samples for a whole plate should not exceed 30 minutes.
   4. Cover the plate with the Plate Seal and incubate at 4°C for 16-18 hours. Keep still, plate rotator not needed for this step.
   5. Incubate plate for 40 minutes at room temperature. Plate rotator not needed for this step.
   6. Remove the Plate Seal and aspirate the solution in the wells. Wash the wells four times with approximately 0.3 mL/well of diluted Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
   7. Pipette 100 µL of SA-HRP Solution into each of the wells.
   8. Cover the plate with a Plate Seal and incubate at room temperature for 2 hours. During the incubation, the plate should be rotated on a plate rotator (~ 100 rpm).
   9. Remove the Plate Seal, aspirate and wash the wells four times with approximately 0.3 mL/well of diluted Wash Solution. Finally, invert the plate and tap it onto an absorbent surface, such as paper toweling, to ensure blotting free of most residual washing solution.
   10. Add 100 µL of Substrate Solution into the wells, cover the plate with a Plate Seal and incubate for 30 minutes at room temperature. Plate rotator not needed for this step.
   11. Add 100 µL of Stop Solution into the wells to stop the reaction.
   12. Read the optical absorbance of the wells at 450 nm. The optical absorbance of reaction solution in wells should be read as soon as possible after stopping the color reaction. Note: Test all samples in duplicate.

The dose-response curve of this assay fits best to a 4 or 5-parameter logistic equation. The results of unkonwn samples can be calculated with any computer program having a 4 or 5-parameter logistic function. Otherwise calculate mean absorbance values of wells containing calibrators and plot a calibration curve on semi logarithmic graph paper (abscissa: concentation of calibrator, ordinate, absorbance values). Use the average absorbance of each sample to determine the corresponding value by simple interpolation from this calibration curve. When a sample value exceeds 100 ng/mL, it must be diluted with Buffer Solution and re-assayed until the sample value is within the assay range.

Notes
1. EDTA-2Na (1 mg/mL) additive blood collection tube is recommended for the plasma collection. Serum and plasma samples must be used as soon as possible after collection. If samples are tested later, they should be aliquoted and frozen at or below -30°C.
2. Calibrator and labeled antigen solutions should be prepared immediately before use. This kit can be used dividedly in strips of the plate. In such case, the rest of reconstituted reagents should be stored at or below -30°C (stable for 1 month).
3. The total pipetting time of calibrator solutions and samples for the whole plate should not exceed 30 minutes.
4. During storage of washing solution (concentrated) at 4°C, precipitates may be observed, however they will be dissolved when diluted. Diluted wash solution is stable for 6 months at 4°C.
5. Pipetting operations may affect the precision of the assay, so pipette all solutions precisely. In addition, use clean test tubes or vessels in assay and use a new tip for each calibrator or sample to avoid cross contamination.
6. When sample concentration exceeds 100 ng/mL, it needs to be diluted with buffer solution to proper concentration.
7. During the incubation with SA-HRP solution at room temperature, the assay plate should be shaken gently with a plate shaker to promote immunoreaction (approximately 100 rpm).
8. Perform all the determination in duplicate.
9. Read plate optical absorbance in wells as soon as possible after adding the stop solution.
10. To quantitate accurately, always run a calibration curve when testing samples.
11. Protect reagents from strong light (e.g. direct sunlight) during storage and assay.
12. Do not mix lots.
13. Some reagents contain human serum (tested and found negative for HBsAG, HIV ﾽ, HCV, HIV-1 AG or HIV-1 NAT, ALT and Syphilis by FDA approved methods), care should be taken when handling.