|
Assay Time
|
4 h
|
|
Plate
|
Pre-coated
|
|
Protocol
|
- Step 1. Add 50?μL of Standard or Sample per well. Incubate 2 hours.
- Step 2. Wash, then add 50?μL of Biotinylated Antibody per well. Incubate 1 hour.
- Step 3. Wash, then add 50?μL of SP Conjugate per well. Incubate 30?minutes.
- Step 4. Wash, then add 50?μL of Chromogen Substrate per well. Incubate 25?minutes.
- Step 5. Add 50?μL of Stop Solution per well. Read at 450 nm immediately.
|
|
Reagent Preparation
|
Freshly dilute all reagents and bring all reagents to room temperature before use. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIX Diluent Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8?°C. Human Growth Hormone Standard: Reconstitute the Human Growth Hormone Standard (1.6?ng) with 1?mL of MIX Diluent to generate a 1.6?ng/mL standard stock solution. Allow the standard to sit for 10?minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution (1.6?ng/mL) 2-fold with MIX Diluent to produce 0.8, 0.4, 0.2, 0.1, 0.05, and 0.025?ng/mL solutions. MIX Diluent serves as the zero standard (0?ng/mL). Aliquot remaining stock solution to limit repeated freeze-thaw cycles. This solution should be stored at -20?°C and used within 48 hours. 4 Standard Point Dilution [GH] (ng/mL) P1 1 part Standard (1.6?ng/mL) 1.6 P2 1 part P1 + 1 part MIX Diluent 0.8 P3 1 part P2 + 1 part MIX Diluent 0.4 P4 1 part P3 + 1 part MIX Diluent 0.2 P5 1 part P4 + 1 part MIX Diluent 0.1 P6 1 part P5 + 1 part MIX Diluent 0.05 P7 1 part P6 + 1 part MIX Diluent 0.025 P8 MIX Diluent 0.0 Biotinylated Human Growth Hormone Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 50-fold with MIX Diluent. The undiluted antibody should be stored at -20?°C. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent. The undiluted conjugate should be stored at -20?°C.
|
|
Sample Collection
|
Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10?minutes and collect plasma. The sample is suggested for use at 1x or within the range of 1x - 4x into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20?°C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10?minutes and remove serum. The sample is suggested for use at 1x or within the range of 1x - 4x into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. The undiluted samples can be stored at -20?°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10?minutes at 4?°C to remove debris and collect supernatants. Samples can be stored at -20?°C or below. Avoid repeated freeze-thaw cycles.
|
|
Assay Procedure
|
Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25?°C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human Growth Hormone Standard or sample to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human Growth Hormone Antibody to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 1 hour. Wash the microplate as described above. 5 Add 50 l of Streptavidin-Peroxidase Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 30?minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 l of Chromogen Substrate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 25?minutes or until the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10?minutes, which will reduce the readings.
|
|
Calculation of Results
|
- Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
- To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
- Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.
|
|
Restrictions
|
For Research Use only
|
