Sample Collection, Preparation, and Storage

1. Serum: After placing whole blood samples at room temperature for 2 hours or at 4°C overnight, centrifuge at 1000×g for 20 minutes. Remove the supernatant for testing. Blood collection tubes should be disposable, pyrogen-free, and endotoxin-free. Store at -20°C or -80°C and avoid repeated freezing and thawing.

2. Plasma: Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing. EDTA-Na₂ is recommended as an anticoagulant. Avoid using samples with hemolysis or hyperlipidemia. Store at -20°C or -80°C and avoid repeated freezing and thawing. 3. Tissue Homogenization: Take an appropriate amount of tissue and wash it in pre-chilled PBS (0.01M, pH 7.0-7.2) to remove blood (lysed red blood cells in the homogenate will affect the measurement results). After weighing, mince the tissue and mix it with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio; the specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS). Pour the mixture into a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or freeze-thawed repeatedly (keep the sonication in an ice bath and repeat the freeze-thaw cycle twice). Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes. Remove the supernatant for analysis. (The tissue homogenate should also be assayed for protein concentration to obtain a more accurate concentration of the test substance per milligram of protein.) 4. Cell Culture Supernatant: Centrifuge the cell supernatant at 1000 × g for 20 minutes to remove impurities and cell debris. Remove the supernatant for testing and store at -20°C or -80°C, but avoid repeated freezing and thawing.

5. Urine: Collect the first morning urine (midstream) or 24-hour urine, centrifuge at 2000×g for 15 minutes, collect the supernatant, and store the sample at -20°C. Avoid repeated freezing and thawing.

6. Saliva: Collect the sample using a saliva sample collection tube, then centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing, or aliquot and store at -20°C. Avoid repeated freezing and thawing.

7. Other biological samples: Centrifuge at 1000×g for 20 minutes, remove the supernatant, and store at -20°C. Avoid repeated freezing and thawing.

Precautions

1. The sample should be clear and transparent, and suspended matter 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 to be tested within one week of collection, it can be stored at 4°C. If testing cannot be done promptly, aliquot the sample into a single-use amount and freeze at -20°C (for testing within one month) or -80°C (for testing within three to six months). Avoid repeated freeze-thaw cycles. Bring the sample to room temperature before the experiment.

Sample Dilution Guidelines

If your test sample requires dilution, the general dilution guidelines are as follows:

1. 50-fold dilution: One-step dilution. Dispense 5 μL of sample into 245 μL of Standard and Sample Diluent for a 50-fold dilution.

2. 100-fold dilution: One-step dilution. 3. 1000-fold dilution: Two-step dilution. Add 5 μL of sample to 95 μL of standard and sample diluent for a 20-fold dilution. Then, add 5 μL of the 20-fold diluted sample to 245 μL of standard and sample diluent for a 50-fold dilution, for a total of 1000-fold dilution. 4. 100,000-fold dilution: Three-step dilution. Add 5 μL of sample to 195 μL of Standard & Sample Diluent for a 40-fold dilution. Then, add 5 μL of the 40-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution. Finally, add 5 μL of the 2,000-fold diluted sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution, for a total dilution of 100,000-fold. 5. For each dilution step, use at least 3 μL of liquid, and the dilution factor should not exceed 100. Excessively small sample volumes can easily lead to greater errors during mixing. Ensure thorough mixing at each dilution step to avoid foaming.

6. If the dilution factor is very high, you can dilute it with PBS first, and then use the standard and sample diluent provided in the kit as the final step.

Sample Dilution Recommendations

1. Normal fresh serum/plasma samples are recommended for testing (Original solution).

2. Due to individual differences, the recommended dilution factor is for reference only. For actual testing, please estimate the sample concentration range in advance and determine the dilution factor of the sample to be tested through preliminary experiments.

Preparation for Testing

1. Please remove the kit from the refrigerator 30 minutes in advance and equilibrate to room temperature.

2. Dilute the 25× concentrated wash buffer to 1× working solution with double-distilled water. Return the unused portion to 4°C. 3. Standards: Add 1.0 mL of Universal Standard & Sample Diluent to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Then gently mix (concentration: 100 ng/mL). Then, serially dilute the standard to 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.13 ng/mL, and 1.57 ng/mL. Use a blank well with the standard diluent (0 ng/mL). Prepare the required amount of standard and set aside. It is recommended that the prepared standard be added to the sample within 15 minutes; it is not recommended to leave it for extended periods. 4. Biotinylated Antibody Working Solution: Before the experiment, calculate the required amount of biotinylated antibody working solution (based on 100 μL/well, the actual amount should be 100-200 μL more). 15 minutes before use, dilute the concentrated biotinylated antibody (1:100) with biotinylated antibody diluent to the working concentration and use it on the same day. The dilution principle is to add 1 μL of concentrated biotinylated antibody to 99 μL of biotinylated antibody diluent and mix thoroughly with a pipette.

5. Enzyme Conjugate Working Solution: Before the experiment, calculate the required volume for the experiment (based on 100 μL/well; add 100-200 μL more). 15 minutes before use, dilute the concentrated HRP enzyme conjugate (1:100) with enzyme conjugate diluent to the working concentration for use that day. The dilution principle is to add 1 μL of concentrated enzyme conjugate to 99 μL of enzyme conjugate diluent and mix thoroughly with a pipette.

6. TMB Substrate - Use a pipette to aspirate the required volume of solution. Do not pour any remaining solution back into the reagent bottle.

Procedure

1. Before beginning the experiment, all reagents should be equilibrated to room temperature. Prepare all reagents in advance. When diluting reagents or samples, mix thoroughly, avoiding foaming as much as possible. If the sample concentration is too high, dilute with sample diluent to bring the sample within the detection range of the kit.

2. Add 100 μL of the standard or sample to be tested (if the sample requires dilution, refer to the sample dilution guidelines for dilution methods). Be careful not to create bubbles. Add the sample to the bottom of the ELISA plate well, avoiding contact with the well walls. Gently shake to mix. Cover the plate or apply film, and incubate at 37°C for 80 minutes. To ensure the validity of the experimental results, use a fresh standard solution for each experiment.

3. Discard the liquid in the wells, spin dry, and wash the plate three times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and discard the liquid from the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 4. Add 100 μL of biotin antibody working solution to each well (can be prepared 15 minutes in advance), cover the plate with film, and incubate at 37°C for 50 minutes. 5. Discard the liquid from the wells and wash the plate three times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and discard the liquid from the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 6. Add 100 μL 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 wells and wash the plate 5 times. Wash each well with 200 μL of washing solution, soak for 1-2 minutes, and shake off the liquid in the ELISA plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 8. Add 90 μL of TMB chromogenic substrate solution to each well and incubate at 37°C in the dark for 20 minutes (shorten or extend the time as appropriate based on the actual color development, but not exceed 30 minutes). 9. Add 50 μL of stop solution to each well to terminate the reaction (the blue color will immediately turn yellow). The order of adding the stop solution should be as similar as possible to the order of adding the color developer. To ensure the accuracy of the experimental results, the stop solution should be added as soon as possible after the substrate reaction time expires. 10. Immediately measure the optical density (OD) of each well using a microplate reader at a wavelength of 450 nm. The instrument should be preheated and the assay program set before use. Calculation of Results 1. Subtract the OD value of the blank well from the OD value of each standard and sample. If replicates are used, the average value should be used for calculation. 2. For ease of calculation, although concentration is the independent variable and OD value is the dependent variable, we use the OD value of the standard as the horizontal axis (X-axis) and the concentration of the standard as the vertical axis (Y-axis) when plotting. To ensure intuitive visualization of the experimental results, the graphs show raw data rather than logarithmic values. Due to differences in experimental operating conditions (such as operator, pipetting technique, plate washing technique, and temperature), the OD values of the standard curve will vary. The provided standard curve is for reference only; experimenters should establish their own standard curve. The sample concentration can be calculated from the OD value of the sample used on the standard curve. This value is then multiplied by the dilution factor to determine the actual sample concentration. It is recommended to use professional curve drawing software, such as Curve Expert.

Note: This picture is for reference only

• Recovery Rate

Recovery rate experiments were performed by adding human GAPDH at known concentrations to different samples to obtain the range and average recovery rate.

4. Samples should be added quickly, and each addition should be controlled within 10 minutes. In order to ensure the accuracy of the experiment, it is recommended to use duplicate wells. When pipetting reagents, keep the addition order consistent from one well to another. This will ensure that the incubation time of all wells is the same.

5. During the washing process, the washing solution remaining in the reaction well should be patted dry on absorbent paper. Do not place the filter paper directly into the reaction well to absorb water. Before reading, be sure to remove the residual liquid and fingerprints at the bottom to avoid affecting the reading of the microplate reader.

6. The color developer TMB should be avoided from 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 already obvious, please stop the reaction in advance to avoid the color being too dark and affecting the reading of the microplate reader.

7. The test tubes and reagents used in the experiment are disposable. Reuse is strictly prohibited, otherwise it will affect the experimental results.

8. Please wear a lab coat and latex gloves during the experiment to ensure proper protection, especially when testing blood or other body fluid samples. Please follow the National Biological Laboratory Safety Protection Regulations.

9. Components of kits with different batches cannot be mixed (except for washing solution and reaction stop solution).

10. The enzyme label strips in the kit are detachable. Please use them in batches according to experimental needs.