Chromogranin A ELISA 试剂盒

Note: This kit can be used dividedly in strips of the plate. In such case, the rest of reconstituted reagents (calibrator, labeled antigen and specific antibody) should be stored below -30°C.
1. Preparation of Buffer solution: Dilute buffer solution (concentrated) (10 mL) to 50 mL with distilled water.
2. Preparation of Calibrator: Reconstitute the lyophilized CgA calibrator (100 pmol) with 1 mL of buffer solution, which affords a 100 pmol/mL calibrator solution. 0.1 mL of the reconstituted calibrator solution is diluted with 0.2 mL of buffer solution to yield an initial calibrator solution of 33.33 pmol/mL. 0.1 mL of the 33.33 pmol/mL calibrator solution is diluted with 0.2 mL buffer solution to make a 11.11 pmol/mL calibrator solution. Repeat the dilution to make each calibrator solution of 3.70, 1.23, 0.41 and 0.14 pmol/mL. Buffer solution is used as 0 pmol/mL. Note: Calibrator Solution must be prepared immediately before assay. Use clean test tubes or vessels.
3. Preparation of Labeled Antigen solution: Dissolve lyophilized labeled antigen with distilled water (6 mL). Note: Labeled Antigen solution must be prepared immediately before assay.
4. Preparation of Specific Antibody solution: Dissolve lyophilized specific antibody with distilled water (12 mL). Note: Specific Antibody solution must be prepared immediately before assay.
5. Preparation of Substrate Solution: Dissolve one OPD Tablet in 12 mL of Substrate Buffer. Note: Substrate Solution must be prepared immediately before assay. Use clean test tubes or vessels.
6. Preparation of Wash Solution: Dilute 50 mL of Wash Solution Concentrate to 1,000 mL with distilled water. Diluted Wash Solution is stable for 3 months at 4°C. Note: During storage of the Wash Solution Concentrate at 4°C, precipitates may be observed, however, they will dissolve when diluted.
7. Other reagents are ready for use.

1. Warm the reagents and samples to room temperature (20-30°C) before beginning the test.
   2. Add 0.35 mL/well of washing solution into the wells and aspirate the washing solution in the wells. Repeat this washing procedure further twice (total three times).
   3. Fill 50 µL of buffer solution into wells first and then introduce 25 µL of calibrator solutions (0, 0.14, 0.41, 1.23, 3.70, 11.11, 33.33 pmol/mL) or samples and add 50 µL of labeled antigen solution, finally add 100 µL of specific antibody solution.
   4. Cover the plate with a Plate Seal and incubate it at room temperature overnight for 16~20 hours with gentle shaking on a microtiter plate shaker. During incubation except color reaction, the test plate should be shaken gently by plate shaker to promote immunoreaction.
   5. Take off the Plate Seal, aspirate and wash the wells three times with approximately 0.35 mL/well of washing solution.
   6. Pipette 100 µL of SA-HRP solution into the wells.
   7. Cover the plate with a Plate Seal and incubate it for two hours at room temperature. During the incubation, the plate should be shaken with a microtiter plate shaker.
   8. Resolve OPD tablet with 12 mL of substrate buffer. It should be prepared immediately before use.
   9. Take off the Plate Seal, aspirate the solution in the wells and wash the wells four times with approximately 0.35 mL/well of washing solution.
   10. Pipette 100 µL of substrate solution into the wells, incubate the plate for 30 minutes at room temperature.
   11. Add 100 µL of stop solution into the wells to stop color reaction.
   12. Read the optical absorbance of the wells at 490 nm. Read optical absorbance of reaction solution in wells as soon as possible after stopping the color reaction. Note: Perform all determinations in duplicate.

Calculate mean absorbance values of wells containing the Calibrators and plot a calibration curve on semilogarithmic graph paper (abscissa: concentration of Calibrators, ordinate: absorbance values of Calibrators). Use the calibration curve to read CgA concentrations in samples from the corresponding absorbance values. When a sample value exceeds 33.33 pmol/mL, it needs to be diluted with buffer solution until the value is within the assay range.