Sku Size Price Quantity EK710989 96 Wells $380
Human growth hormone (HGH) Elisa Kit quantity
MSDS
Product Information Components Test Principal Sample Collection Reagent Preparation Assay Procedure Troubleshooting
SKU: EK710989 Categories: Elisa Kits, Sandwich Elisa Kit Species: Human Assay Principle: Quantitative Assay Type: Sandwich Target Name: growth hormone (HGH) Sample Type: Serum, Plasma, Urine, Tissue Homogenates, Cell Culture Supernates Sensitivity: 15 pg/ml Detection Range Low: 48 pg/ml Detection Range High: 2800 pg/ml Standard: 3600 pg/ml Assay Time: 1-5 h Sample Volume: 50 - 100 μL Detection Wavelength: 450 nm Research Area: Signal Transduction Abbreviation: HGH
Components
Components Quantity 1 x 96 Tests Storage Condition Pre-Coated Microplate 8 wells ×12 strips 4°C/-20°C Standard (Liquid) 1×0.5mL 4°C/-20°C Standard Diluent 1×1.5mL 4°C/-20°C HRP-Conjugate Reagent 1×6mL 4°C/-20°C Sample Diluent 1×6mL 4°C/-20°C Chromogen Solution A 1×6mL 4°C/-20°C Chromogen Solution B 1×6mL 4°C/-20°C Stop Solution 1×6mL 4°C/-20°C Wash Solution 1×20ml×30 fold 4°C/-20°C User manual 1 pieces Adhesive Strip 2 pieces
Test Principle
This ELISA kit employs a quantitative enzyme-linked immunosorbent assay (ELISA) method. The micro-ELISA strip plate provided in this kit has been pre-coated with an antibody specific to growth hormone (HGH). Standards or samples are added to the appropriate wells of the micro-ELISA strip plate, where they bind to the specific antibody. Subsequently, a horseradish peroxidase (HRP)-conjugated antibody specific for growth hormone (HGH) is added to each well and incubated. Any unbound components are washed away. The TMB substrate solution is then added to each well. Only those wells containing growth hormone (HGH) and the HRP-conjugated antibody will develop a blue color, which subsequently turns yellow upon the addition of the stop solution. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm. The OD value is proportional to the concentration of growth hormone (HGH) present. The concentration of growth hormone (HGH) in the samples can be calculated by comparing the OD of the samples to a standard curve.
Sample Collection
Serum: Allow samples to clot for 1 hour at room temperature or overnight at 2–8°C before centrifugation at 1,000×g for 20 minutes at 2–8°C. Collect the supernatant to carry out the assay.
Plasma: Collect plasma using EDTA-Na2 as an anticoagulant. Centrifuge samples at 1,000×g for 15 minutes at 2–8°C within 30 minutes of collection. Collect the supernatant to carry out the assay.
Tissue Homogenates: It is recommended to consult specific literature for different tissue types before processing. Note that hemolyzed blood may interfere with results; therefore, tissues should be minced into small pieces and rinsed thoroughly in ice-cold PBS (0.01M, pH 7.4) to remove excess blood. Weigh the tissue pieces and homogenize them in PBS at a ratio of 1:9 (e.g., 1g tissue to 9mL PBS) using a glass homogenizer on ice. To further disrupt the cells, sonicate the suspension with an ultrasonic cell disrupter or subject it to repeated freeze-thaw cycles. Centrifuge the homogenates at 5,000×g for 5–10 minutes at 2–8°C and collect the supernatant.
Cell Lysates: For adherent cells, gently wash the cells with a moderate amount of pre-cooled PBS and dissociate them using trypsin. Collect the cell suspension into a centrifuge tube and centrifuge at 1,000×g for 5 minutes. Discard the medium and wash the cells three times with pre-cooled PBS. Resuspend the cells in pre-cooled PBS (approximately 150–250 μL of PBS per 1×106 cells). Lyse the cells via repeated freeze-thaw cycles or ultrasonic cell disruption until the cells are fully lysed. Centrifuge at 1,500×g for 10 minutes at 2–8°C. Remove cell debris and collect the supernatant for the assay.
Urine: Collect the first midstream urine of the day directly into a sterile container. Centrifuge to remove particulate matter. Assay immediately or store in aliquots at ≤ −20°C. Avoid repeated freeze-thaw cycles.
Saliva: Collect saliva using a specialized collection device. Centrifuge the samples at 1,000×g for 15 minutes at 2–8°C. Remove any remaining particulates and assay immediately, or store in aliquots at ≤ −20°C. Avoid repeated freeze-thaw cycles.
Feces: Collect dry feces, ideally weighing more than 50 mg. Dilute the sample with PBS or lysis buffer at a ratio of 1:9 (w:v) (e.g., add 900 μL of buffer to 100 mg of feces). Homogenize the sample by mashing or sonication. Centrifuge the mixture at 5,000×g for 10 minutes and collect the supernatant for testing.
Cell Culture Supernatant and Other Biological Fluids: Centrifuge samples at 1,000×g for 20 minutes at 2–8°C. Collect the supernatant to carry out the assay.
Recommended Reagents for Sample Preparation: 10× EDTA Anticoagulant, PMSF Protease Inhibitor, 0.25% Trypsin Solution.
Notes
Samples intended for use within 5 days may be stored at 4°C; otherwise, samples must be stored at −20°C (for up to 1 month) or −80°C (for up to 2 months) to avoid loss of bioactivity and contamination. Sample hemolysis can influence the results, so hemolytic specimens should not be used. When performing the assay, bring the samples to room temperature.
Reagent Preparation
Prepare Wash Buffer: Combine 20 mL of concentrated wash buffer (the entire bottle) with 580 mL of deionized (DI) water.
Standard Preparation: Prepare the standard series by pipetting 100 μL of standard diluent into the first four tubes. Pipette 200 μL of the stock standard into the fifth tube. Perform a serial dilution by transferring 200 μL from the fifth tube into the fourth tube and mixing thoroughly. Next, transfer 100 μL from the fourth tube into the third tube, continuing the series as described below. The undiluted stock serves as the high standard, while the sample diluent serves as the zero standard (blank).
Tube 6 (Stock) 5 4 3 2 1 0 (Blank) pg/ml 3600 2400 1600 800 400 200 0
Assay Procedure
Prepare Standards:
Label six microcentrifuge tubes (5 down to 0). Pipette 100 μL of Standard Diluent into tubes 4, 3, 2, 1, and 0. Pipette 200 μL of the Stock Standard into tube 5. Perform a serial dilution: Transfer 100 μL from tube 5 into tube 4 and vortex to ensure homogeneity. Continue the serial dilution by transferring 100 μL from tube 4 into tube 3, and repeat this process sequentially until tube 1 is prepared. Tube 0 (Reagent Blank): This tube should contain only 100 μL of Standard Diluent to serve as the zero concentration point. Add Standards: Add 50 μL of each diluted standard (Tubes 0–5) in duplicate into the designated wells of the 96-well microplate. Add Sample Diluent: Using a multichannel pipette, dispense 40 μL of Sample Diluent into all wells designated for test samples. Note: Do not add diluent to wells already containing standards. Sample Loading and First Incubation: Add 10 μL of each test sample into the designated wells in duplicate. Mix gently by tapping or using a plate shaker. Seal the plate with an adhesive strip and incubate at 37°C for 40 minutes. Wash the Plate: Aspirate the liquid from all wells. Wash the plate five times using 300 μL of Wash Buffer per well. Ensure all residual liquid is removed by blotting the plate on absorbent paper. HRP-Conjugate Addition and Second Incubation: Dispense 50 μL of HRP-Conjugate reagent into every well, including the standard wells but excluding the blank well. Seal the plate and incubate at 37°C for 40 minutes. Second Washing: Repeat the wash procedure as described in Step 5 (five cycles of 300 μL). Chromogenesis (Color Development): Add 50 μL of Chromogen Solution A followed by 50 μL of Chromogen Solution B to each well. Mix gently. Protect the plate from light. Incubate at 37°C for 20 minutes. Reaction Termination: Add 50 μL of Stop Solution to each well. The color should shift from blue to yellow. Measurement: Measure the optical density (OD) at 450 nm using a microplate reader immediately (within 15 minutes of adding the stop solution).
Calculation of Results
Plot the standard concentration on the horizontal axis and the OD values on the vertical axis to create the standard curve. Locate the sample OD on the standard curve and find the associated concentration, then multiply by the dilution factor. Alternatively, calculate the linear regression equation of the standard curve and substitute the sample OD to calculate the concentration.
Troubleshoot Common Elisa Issues
Pipetting & Washing Color Signal High Background Poor Standard Curve Poor Replicate Data Assay Variable
Pipetting and washing techniques for ELISA
Pipetting technique
Use the correct pipette that is within the range suggested by manufacturer Confirm tip is firmly seated on the pipette Confirm there are no air bubbles while pipetting Change tips between each standard, sample, or reagent Use different reservoirs for each reagent Pipette sample into the side of wells to avoid splashing Always run samples/standards in replicate
Washing Procedure
Completely aspirate liquid from all wells by gently lowering an aspiration tip into the bottom of each well. Note: Take care not to scratch the inside of the well. Fill the wells with at least 500 μL of diluted wash buffer Let soak for 25 to 30 seconds Aspirate wash buffer from wells Repeat as directed in protocol (usually 4-5 times) After washing is complete, invert plate and tap (forcefully, if necessary) dry on absorbent tissue. Be sure to remove any residual liquid. Alternatively, a squirt bottle or automated plate washer may be used. Be sure to follow the above.
Problem: No color plate
Possible Cause Solution Mixed use of component reagents Please read labels clearly when preparing or using In the process of plate washing and sample addition, the enzyme marker is contaminated and inactivated, and loses its ability to catalyze the color developing agent Confirm that the container holding the ELISA plate does not contain enzyme inhibitors (such as NaN3, etc.), and confirm that the container for preparing the Wash Solution has been washed. Missing a reagent or a step Review the manual in detail and strictly follow the operating steps.
Problem: Light Color
Possible Cause Solution The sample uses NaN3 preservative, which inhibits the reaction of the enzyme Samples cannot use NaN3 The sample to be tested may not contain strong positive samples, so the result may be normal In case of doubt, please test again. Wrong filter used for absorbance reading When TMB is used as the substrate, the absorbance should be read at 450 nm. Insufficient incubation time Make sure to follow recommended incubation times. Insufficient color reaction Usually 15 - 30 minutes The number of washings increases, and the dilution ratio of the concentrated lotion does not meet the requirements Reduce the impact of washing, dilute the concentrated lotion and washing time according to the manual, and accurately record the washing times and dosage. Distilled water quality problem The prepared lotion must be tested to see if the pH value is neutral. In the process of plate washing and sample addition, the enzyme marker is contaminated and inactivated, and loses its ability to catalyze the color developing agent. Confirm that the container holding the ELISA plate does not contain enzyme inhibitors (such as NaN3, etc.), confirm that the container for preparing the Washing Solution has been washed, and confirm that the purified water for preparing the Washing Solution meets the requirements and is not contaminated. The kit has expired or been improperly stored Please use it within the expiration and store it in accordance with the storage conditions recommended in the manual to avoid contamination. Reagents and samples are not equilibrated before use All reagents and samples should be equilibrated at room temperature for about 30 minutes. Insufficient suction volume of the pipette, too fast discharge of pipetting suction, too much liquid hanging on the inner wall of the tip or the inner wall is not clean. To calibrate the pipette, the tips should be matched, each time the tips should fit tightly, the pipetting should not be too fast, and the discharge should be complete. The inner wall of the tips should be clean, and it is best to use it once. Incubation temperature constant temperature effect is not good Keep the temperature constant to avoid the local temperature being too high or too low When adding liquid, too much remains on the medial wall of wells When adding liquid, the tip should try to add liquid along the bottom of the medial wall of wells without touching the bottom of the hole. Reuse of consumables The tips should be replaced when different reagents are drawn, and different storage vessels should be used when configuring different reagent components. The bottom of the microwell is scratched or there is dirt During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other. Cross-contamination during sample addition Try to avoid cross-contamination when adding samples Cross-contamination from manual plate washing When washing the plates by hand, the first 3 injections of the lotion should be discarded immediately, and the soaking time should be set for the next few times to reduce cross-contamination. Cross-contamination when clapping Use a suitable absorbent paper towel when clapping the plate, do not pat irrelevant substances into the well of the plate, and try not to pat in the same position to avoid cross-contamination. The liquid filling head of the plate washer is blocked, resulting in unsatisfactory liquid addition or large residual amount of liquid suction, resulting in the color of plate is chaotic and irregular. Unblock the liquid addition head, so that each well is filled with washing liquid when washing the plate and the residual amount should be small when aspirating liquid. Incomplete centrifugation of the sample, resulting in coagulation in the reaction well or interference of sediment or residual cellular components Serum plasma should be fully centrifuged at 3000 rpm for more than 6 minutes The sample is stored for too long time, resulting in contamination. Samples should be kept fresh or stored at low temperature to prevent contamination Incorrect preparation of Washing Solution or direct misuse of concentrated Washing Solution Please configure according to the manual
Weak or no signal in ELISA
Possible Cause Solution Reagents not at room temperature at start of assay It is recommended that all reagents be at room temperature before starting the assay. Allow reagents to sit on bench for 15–20 minutes to reach room temperature. Incorrect storage of components Double check storage conditions on kit label. Most kits need to be stored at 2–8oC. Expired reagents Confirm expiration dates on all reagents. Do not use reagents that are past the expiration date. Reagents added/prepared incorrectly Check protocol, ensure reagents were added in the proper order and prepared to correct dilution. Incorrect dilutions prepared Check pipetting technique—see below—and double check calculations. Capture antibody didn’t bind to plate If using a ready-to-use kit: Manufactured kits come with plates pre-coated with capture antibody. If coating your own plate with an Antibody Pair Kit: Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps. Wells scratched with pipette or washing tips Use caution when dispensing and aspirating into and out of wells. Automated plate washers may need to be calibrated so tips don’t touch bottom of wells. Plate read at incorrect wavelength Make sure to use recommended wavelength/filter.
Too much signal in ELISA
Possible Cause Solution Insufficient washing Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid. Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other. Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations. Longer incubation times thanrecommended Make sure to follow recommended incubation times.
Problem: High background in ELISA
Possible Cause Solution Insufficient washing Use appropriate washing procedure—see below. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid. Substrateexposed to light prior to use Ensure substrate isnot exposed to light—store in a dark place. Limit exposure to light whilerunning assay. The yellowingof the whole plate may be caused by wrong addition of other reagents Check the componentsand lot numbers of the reagents before the experiment, and confirm that allcomponents belong to the corresponding kit. Reagents from differentkits or different lot numbers cannot be mixed. Streptavidin-HRP contaminates the tip and TMB container orpositive control contaminates the Pre-coated Microplate When absorbingdifferent reagents, the tips should be replaced. When configuring differentreagent components, different storage vessels should be used. Please use apipette during operation. BiotinylatedAntibody or Streptavidin-HRP concentration too high Checkwhether the concentration calculation is correct or use after furtherdilution. Color development time is too long Pleasestrictly follow the steps of the manual. Longerincubation times than recommended Make sure to followrecommended incubation times. Incorrectstandard curve dilutions prepared Check pipettingtechnique—see below—and double-check calculations. The wrongfilter was used when the absorbance value was read When TMB is used asthe substrate, the absorbance should be read at 450 nm.
Problem: High background in ELISA
Possible Cause Solution Incorrect standard curve dilutions prepared Check pipetting technique—see below—and double-check calculations. Capture antibody didn’t bind to plate Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps.
Problem: Poor replicate data in ELISA
Possible Cause Solution Insufficient washing Use appropriate washing procedure—see below. Increasing duration of soak steps may also help. Add 30 seconds each time you let wash buffer soak. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid. Capture antibody didn’t bind to plate Ensure that you are using an ELISA plate, not a tissue culture plate. Dilute antibody in PBS. Ensure correct preparation and incubation time for both coating and blocking steps. Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other.
Problem: Inconsistent results assay-to-assay in ELISA
Possible Cause Solution Insufficient washing Use appropriate washing procedure—see below. At the end of each washing step, invert plate on absorbent tissue and allow to completely drain, tapping forcefully if necessary to remove any residual fluid. Inconsistent incubation temperature Manufactured kits have optimized protocols. Make sure to follow recommended incubation temperatures. Be aware of fluctuations in temperature due to environmental conditions. Plate sealers not used or reused During incubations, cover assay plates with plate sealers. Use a fresh sealer each time the plate is opened. This will prevent wells from contaminating each other. Incorrect dilutions prepared Check pipetting technique—see below—and double-check calculations.