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RAT/MOUSE GROWTH HORMONE ELISA KIT

互联网2013-11-13

2091

实验原理

This assay is a Sandwich ELISA based, sequentially, on: 1) capture of rat or mouse Growth Hormone molecules from samples to the wells of a microtiter plate coated by a pre-titered amount of anti-Growth Hormone polyclonal antibodies, 2) wash away of unbound materials from samples, 3) binding of a second biotinylated anti-Growth Hormone polyclonal antibody to the captured molecules, 4) wash away of unbound materials from samples, 5) conjugation of horseradish peroxidase to the immobilized biotinylated antibodies, 6) wash away of free enzyme conjugates, and 7) quantification of immobilized antibody-enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the substrate 3,3’,5,5’-tetramethylbenzidine. The enzyme activity is measured spectrophotometrically by the increased absorbency at 450 nm, corrected from the absorbency at 590 nm, after acidification of formed products. Since the increase in absorbency is directly proportional to the amount of captured rat or mouse Growth Hormone in the unknown sample, the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of Rat Growth Hormone.

实验试剂

Pipettes and Pipette Tips: 10 µL - 20 µL or 20 µL - 100 µL

Multi-Channel Pipettes and Pipette Tips: 5 ~ 50 µL and 50 ~ 300 µL

Buffer and Reagent Reservoirs

Deionized Water

Microtiter Plate Reader capable of reading absorbency at 450 nm

Orbital Microtiter Plate Shaker

Absorbent Paper or Cloth

实验步骤

1. SAMPLE PREPARATION

1) No dilution or preparation is needed for normal serum or plasma samples. In the event that any sample is above 50 ng/mL range, dilutions should be performed using the Assay Buffer provided.

2) Tissue extracts or cell culture samples may require dilution. Dilutions should be performed using the Assay Buffer provided.

2. Pre-warm all reagents to room temperature prior to setting up the assay.

1) Dilute the 10X concentrated Wash Buffer 10 fold by mixing the entire contents of both buffer bottles with 900 mL deionized or distilled water.

2) Remove the required number of strips from the Microtiter Assay Plate. Unused strips should be resealed in the foil pouch and stored at 2-8 °C. Assemble the strips in an empty plate holder and wash well with 300 µl of diluted Wash Buffer. Incubate at room temperature for 5 minutes. Decant Wash Buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times. Do not let wells dry before proceeding to the next step. If an automated machine is used for the assay, use a gentle wash program for all washing steps described in this protocol.

3) Add in duplicate, 100 µL Assay Buffer to the blank wells.

4) Add in duplicate, 90 µL Assay Buffer to Standard wells, QC1, QC2, and sample wells.Add in duplicate, 10 µL Rat/Mouse Growth Hormone Standards in the order of ascending concentration to the appropriate wells. Add in duplicate, 10 µL QC1 and 10 µL QC2 to the appropriate wells. Add sequentially, 10 µL of the unknown samples in duplicate to the remaining wells. For best result all additions should be completed within 30 minutes

5) Cover the plate with plate sealer and incubate at room temperature for 1.5 hours on an orbital microtiter plate shaker set to rotate at moderate speed, approximately 400 to 500 rpm.

6) Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells.

7) Wash wells 3 times with diluted Wash Buffer, 300 µL per well per wash. Decant and tap firmly after each wash to remove residual buffer.

8) Add 100 µL Detection Antibody to all wells. Cover the plate with plate sealer and incubate at room temperature for 1 hour on an orbital microtiter plate shaker set to rotate at moderate speed, approximately 400 to 500 rpm.

9) Remove plate sealer and decant solutions from the plate. Tap as before to remove residual solutions in the wells.

10) Wash wells 3 times with diluted Wash Buffer, 300 µL per well per wash. Decant and tap firmly after each wash to remove residual buffer.

11) Add 100 µL Enzyme Solution to each well. Cover plate with sealer and incubate with moderate shaking at room temperature for 30 minutes on the microtiter plate shaker.

12) Remove sealer, decant solutions from the plate, and tap plate to remove the residual fluid.

13) Wash wells 3 times with diluted Wash Buffer, 300 µL per well per wash. Decant and tap firmly after each wash to remove residual buffer.

14) Add 100 µL of Substrate Solution to each well, cover plate with sealer and shake on the plate shaker for approximately 8 to 15 minutes (A longer development time may be needed if using a plate washer). Blue color should be formed in wells of Growth Hormone standards with intensity proportional to increasing concentrations of Growth Hormone.

15) Remove sealer and add 100 µL Stop Solution [CAUTION: CORROSIVE SOLUTION] and shake plate by hand to ensure complete mixing of solution in all wells. The blue color should turn to yellow after acidification. Read absorbance at 450 nm and 590 nm in a plate reader within 5 minutes and ensure that there are no air bubbles in any well. Record the difference of absorbance units. The absorbance of the highest Growth Hormone standard should be approximately 2.0-3.2, or not to exceed the capability of the plate reader used.

3. INTERPRETATION

1) The assay will be considered accepted when all Quality Control values fall within the calculated Quality Control Range. If any QC’s fall outside the control range, review results with a supervisor.

2) If the difference between duplicate results of a sample is >10% CV, repeat the sample.

3) The limit of sensitivity of this assay is 0.07 ng/mL Rat/Mouse Growth Hormone (10 µL sample size).

4) The appropriate range of this assay is 0.07 ng/mL to 50 ng/mL Rat/Mouse Growth Hormone (10 µL sample size). Any result greater than 50 ng/mL in a 10 µL sample should be diluted using Assay Buffer, and the assay repeated until the results fall within range. Tissue extracts or cell culture media samples greater than 50 ng/mL in a 10 µL sample should be diluted in Assay Buffer.

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