Human PAH ELISA Kit

1.Sample collection preparation and preservation

1. Serum: The whole blood sample is placed at room temperature for 2 hours or 4 °C overnight, and then centrifuged at 1000 × g for 20 minutes, and the supernatant can be detected.
The blood collection tubes shall be disposable non-pyrogenic, non-endotoxin tubes.
Store at-20 °C or-80 °C, avoid repeated freezing and thawing.

2. Plasma: The sample was centrifuged at 2-8 °C and 1000 × g for 15 minutes within 30 minutes after collection, and the supernatant was taken for detection.
EDTA-Na is recommended for anticoagulants₂, avoid using hemolytic, hyperlipidemic samples.
Store at-20 °C or-80 °C, avoid repeated freezing and thawing.

3. Tissue homogenate: Take an appropriate amount of tissue block, wash and remove blood in pre-cooled PBS (0.01 M, pH 7.0-7.2) (the lysed red blood cells in the homogenate will affect the measurement results), cut the tissue after weighing, and then add it to the glass homogenizer with the corresponding volume of PBS (generally according to the mass-volume ratio of 1: 9, the specific volume can be appropriately adjusted according to the experimental needs, and make a record.
It is recommended to add protease inhibitors to PBS).
In order to further lyse tissue cells, the homogenate can be subjected to ultrasonic disruption or repeated freeze-thaw treatment (pay attention to ice bath cooling during ultrasonic disruption, and the repeated freeze-thaw method can be repeated twice).
Finally, centrifuge the prepared homogenate at 5000 × g for 5-10 minutes, and take the supernatant for detection.

4. Cell culture supernatant: Centrifuge the cell supernatant at 1000 × g for 20 minutes to remove impurities and cell debris.
Take the supernatant for detection and store at-20 °C or-80 °C, but avoid repeated freezing and thawing.

5. Urine: Please collect the first urine (middle urine) in the morning, or 24-hour urine, collect the supernatant after centrifugation at 2000 × g for 15 minutes, and store the sample at-20 °C, and avoid repeated freezing and thawing.

6. Saliva: Collect the sample with a saliva sample collection tube, then centrifuge it at 2-8 °C, 1000 × g for 15 minutes, take the supernatant for detection, or store it at-20 °C after packaging.
Avoid repeated freezing and thawing.

7. Other biological samples: Please centrifuge at 1000 × g for 20 minutes, and take the supernatant for detection.

attention：

1. The sample should be clear and transparent, and the suspended solids should be removed by centrifugation.
Hemolysis of the sample will affect the results, so hemolyzed samples should not be used.

2. If the sample is tested within 1 week after collection, it can be stored at 4 °C.
If it cannot be tested in time, please pack it according to the one-time usage amount and freeze it at-20 °C (test within 1 month), or-80 °C (test within 3-6 months) to avoid repeated freezing and thawing.
Keep the sample at room temperature prior to the experiment.

3. If the concentration of the detected substance in your sample is higher than the highest value of the standard product, please dilute it at an appropriate multiple according to the actual situation (it is recommended to do a pre-experiment first to determine the dilution multiple).

Two,Preparation for testing

1. Please take out the kit from the refrigerator 30 minutes in advance and balance it to room temperature.

2. Dilute 25 × concentrated washing solution into 1 × working solution with double distilled water, and put the unused solution back to 4 °C.

3. Standard: Add 1 and 0 mL of standard & sample universal diluent to the freeze-dried standard, tighten the tube cap, and let it stand for 10 minutes.
After it is fully dissolved, gently mix (concentration is 100 ng/mL).
Thereafter, double dilutions were performed to 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.13 ng/mL, 1.57 ng/mL standard dilution (0 ng/mL) as blank wells.
Configure the standard according to the amount you need for later use.
It is recommended to add the configured standard within 15 minutes, and it is not recommended to leave it for too long.

4. Biotinylated antibody working solution: Calculate the dosage required for the current experiment before the experiment (based on 100μL/well, an additional 100-200μL should be configured in actual configuration).
15 minutes before use, use biotinylated antibody diluent Dilute and concentrate biotinylated antibody (1: 100) to a working concentration and use it on the same day.
Dilution principle: Take 1μL of concentrated biotinylated antibody and add it to 99μL of biotinylated antibody diluent, and mix well with a pipette.

5. Enzyme conjugate working solution: Calculate the dosage required for the current experiment before the experiment (based on 100μL/well, an additional 100-200μL should be configured during actual configuration).
Fifteen minutes prior to use, the concentrated HRP enzyme conjugate (1: 100) was diluted with enzyme conjugate diluent to the working concentration and used on the same day.
Dilution principle: Take 1μL of concentrated enzyme conjugate and add it to 99μL of enzyme conjugate diluent, and mix well with a pipette.

6. TMB substrate-use a pipette to draw the required dose of solution, and do not pour the residual solution back into the reagent bottle again.

attention：

1. Please make sure that all components are dissolved and mixed before using the kit.
If the reconstituted standard is not used, please discard it.

2. Concentrate the biotinylated antibody.
The volume of the concentrated enzyme conjugate is small and may be dispersed in various parts of the tube during transportation.
Please centrifuge at 1000 × g for 1 minute before use, so that the liquid on the tube wall or bottle cap can be deposited to the bottom of the tube.
Mix the solution by carefully pipetting 4-5 times with a pipette before taking.
Standard, biotinylated antibody working solution and enzyme conjugate working solution should be prepared according to the required dosage, and the corresponding diluent should be used to prepare without confusion.

3. The concentrated washing liquid taken out of the refrigerator may have crystallization, which is a normal phenomenon.
The crystals can be completely dissolved in a water bath or incubator before arranging the washing liquid (the heating temperature should not exceed 40 °C).
The wash liquid should be at room temperature when used.

4. Sample addition needs to be quick, and it is best to control each sample addition within 10 minutes.
In order to ensure the accuracy of the experiment, it is recommended to use double holes.
Maintaining a consistent sequence of addition from well to well when pipetting reagents will ensure the same incubation time for all wells.

5. During the washing process, the washing liquid remaining in the reaction hole should be patted dry on absorbent paper.
Do not put the filter paper directly into the reaction hole to absorb water.
Before reading, pay attention to removing the residual liquid and fingerprints at the bottom, so as not to affect the reading of the microplate reader.

6. The developer TMB should avoid direct exposure to strong light during storage and use.
After adding the substrate, pay attention to the color change in the reaction well.
If the gradient is obvious, please terminate the reaction in advance to avoid too dark color affecting the reading of the microplate reader.

7. The test tubes and reagents used during the experiment are disposable, and it is strictly forbidden to reuse them, otherwise it will affect the experimental results.

8. Please wear a laboratory coat and latex gloves for protection during the experiment, especially when testing blood or other body fluid samples, please follow the national biological laboratory safety protection regulations.

9. Kit components of different batch numbers cannot be mixed (except washing liquid and reaction termination liquid).

10. The enzyme labeling strip in the kit is a detachable plate, please use it in batches according to the experimental requirements.

Three,Operation steps

1. Before the experiment starts, all reagents should be balanced to room temperature, and all reagents should be configured in advance.
When reagents or samples are diluted, they should be mixed well, and blistering should be avoided as much as possible when mixing well.
If the sample concentration is too high, dilute with a sample diluent to bring the sample within the range of the kit.

2. Add 100μL of the standard product or sample to be tested (if the sample needs to be diluted, please refer to the sample dilution principle for the dilution method).
Be careful not to have bubbles.
When adding the sample, add the sample to the bottom of the well of the labeled plate, try not to touch the well wall, gently shake and mix well, add a cover or film to the labeled plate, and incubate at 37 °C for 80 minutes.
To ensure the validity of the experimental results, please use a new standard solution for each experiment.

3. Discard the liquid in the hole, spin dry, and wash the plate 3 times.
Each well was washed with 200 μL of washing solution, soaked for 1-2 minutes, and the liquid in the labeled plate was removed (or the plate was washed with a plate washer).
After the last wash was complete, the plate was pat dry on absorbent paper.

4. Add 100μL of biotin antibody working solution to each well (can be prepared 15 minutes in advance), coat the enzyme labeled plate, and incubate at 37 °C for 50 minutes.

5. Discard the liquid in the hole and wash the plate 3 times.
Each well was washed with 200 μL of washing solution, soaked for 1-2 minutes, and the liquid in the labeled plate was removed (or the plate was washed with a plate washer).
After the last wash was complete, the plate was pat dry on absorbent paper.

6. Add 100μL of enzyme conjugate working solution to each well (can be prepared 15 minutes in advance), and incubate at 37 °C for 50 minutes.

7. Discard the liquid in the hole and wash the plate 5 times.
Each well was washed with 200 μL of washing solution, soaked for 1-2 minutes, and the liquid in the labeled plate was removed (or the plate was washed with a plate washer).
After the last wash was complete, the plate was pat dry on absorbent paper.

8. Add 90μL of TMB chromogenic substrate solution to each well, and incubate at 37 °C in the dark for 20 minutes (shorten or prolong according to the actual color development, but not exceed 30 minutes).

9. Add 50μL of stop solution to each well to stop the reaction (blue turns yellow immediately at this time).
The sequence of addition of the terminating solution should be the same as that of the developer as possible.
In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.

10. Immediately measure the optical density value (OD value) of each well with a microplate reader at a wavelength of 450 nm.
The instrument should be preheated before use, and the testing program should be set up.

Results Calculation

1. The OD value of each standard and sample should be subtracted from the OD value of the blank well.
If a double hole is set, the average value should be taken for calculation.

2. For the convenience of calculation, although the concentration is the independent variable and the OD value is the dependent variable, we still use the OD value of the standard product as the abscissa (X axis) and the concentration of the standard product as the ordinate (Y axis) when drawing.
At the same time, for the intuition of the test results, the figure provides raw data instead of logarithmic values.
Due to different experimental operating conditions (such as operator, pipetting technology, plate washing technology and temperature conditions, etc.), the OD value of the standard curve will be different.
The standard curve provided is for reference only, and the experimenter needs to establish the standard curve according to his own experiment.
The OD value of the used sample can be calculated on the standard curve to calculate the sample concentration, and then multiplied by the dilution factor, which is the actual concentration of the sample.
It is recommended to use professional curve drawing software such as curve expert.

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• Precision

Intraplate precision (precision within the assay):% CV < 8%

Three samples of known concentrations were tested 20 times each on one plate to assess in-plate precision.

Inter-plate precision (measuring inter-plate precision): CV% < 10%

Three samples of known concentrations were tested 40 times on three different plates to assess the accuracy between the assay plates.

• Recovery

Add known concentration of human PAH to different samples, and do recovery experiment.
The recovery range and average recovery are obtained

• linear

The samples added with human PAH were diluted 2 times, 4 times, 8 times and 16 times for recovery experiments, and the recovery range was obtained