I. 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. The supernatant is then collected 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 your first morning urine (midstream) or 24-hour urine collection. 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, collect the supernatant, and store at -20°C. Avoid repeated freezing and thawing.

Precautions

1. Samples 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. Samples can be stored at 4°C if tested within one week of collection. If testing cannot be performed promptly, aliquot the sample into single-use portions 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 experimenting.

II. Sample Dilution Principles

If your test sample requires dilution, general dilution principles 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. For each dilution step, take at least 3 μL of liquid, and the dilution factor should not exceed 100. Too small a sample volume can easily lead to greater errors during mixing. Ensure that each dilution step is mixed thoroughly to avoid foaming.

6. For very high dilution ratios, dilute with PBS first, and then use the standard and sample diluent provided in the kit as the final step.

III. Pre-Assay Preparation

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

2. Dilute 25× concentrated wash buffer to 1× working solution with double-distilled water. Return the remaining solution to 4°C.

3. Standards: Add 1.0 mL of universal standard and sample diluent to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Then gently mix (concentration is 500 μg/mL). Next, serially dilute the standard solution to 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.63 μg/mL, and 7.82 μg/mL. Use 0 μg/mL as a blank well. Prepare the required amount of standard solution and set aside. It is recommended to add the prepared standard solution to the sample within 15 minutes; do not allow it to sit for an extended period. 4. Biotinylated Antibody Working Solution: Before the experiment, calculate the required amount of biotinylated antibody working solution (calculated as 100 μL/well, and 100-200 μL should be added during the actual preparation). 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 when preparing). 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.

IV. Pre-experimental Preparation

1. Equilibrate all materials and prepared reagents to room temperature before use. Before use, mix all reagents thoroughly, taking care not to produce any foam.

2. Users should calculate the number of samples likely to be used throughout the experiment. Please reserve sufficient sample in advance.

3. Estimate concentrations before measurement. If these values are not within the standard curve range, users must determine the optimal sample dilution for their specific experiment.

V. Operational Overview

2. Discard the liquid from the ELISA plate, add 200 μL of wash buffer to each well, and wash three times. After patting dry, add 100 μL of biotinylated antibody working solution to each well and incubate at 37°C for 50 minutes. 3. Discard the liquid from the ELISA plate, add 200 μL of wash buffer to each well, and wash three times. After patting dry, add 100 μL of HRP enzyme working solution to each well and incubate at 37°C for 50 minutes. 4. Discard the liquid from the ELISA plate, add 200 μL of wash buffer to each well, and wash five times. After patting dry, add 90 μL of TMB to each well and incubate at 37°C for 20 minutes. 5. Add 50 μL of stop solution to each well, read immediately at 450 nm, and calculate the results. 6. Procedure 1. Before beginning the experiment, all reagents should be equilibrated to room temperature and prepared in advance. When diluting reagents or samples, mix thoroughly and avoid foaming. 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 needs to be diluted, 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 sides. Gently shake to mix. Cover the plate or seal with 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, soaking for 1-2 minutes. Spin off the liquid in the plate (or wash with a microplate 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 in 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 in the plate (or use 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 five times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and discard the liquid in the plate (or use 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 depending on the actual color development, but do 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 stop solution should be added in the same order as the developer. To ensure accurate results, add the stop solution 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. Preheat the instrument and set the assay program before use. VII. Calculation of Results 1. Subtract the OD value of the blank well from the OD value of each standard and sample. If replicate wells 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 still 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 drawing the graph. At the same time, for the intuitiveness of the experimental results, the figure provides the original data rather than the logarithmic value. Due to different experimental operating conditions (such as operators, pipetting techniques, plate washing techniques and temperature conditions, etc.), the OD value of the standard curve will vary. The standard curve provided is for reference only, and the experimenter needs to establish a standard curve based on his own experiment. The OD value of the used sample can be used to calculate the sample concentration on the standard curve, and then multiplied by the dilution factor to obtain the actual concentration of the sample. It is recommended to use professional curve drawing software, such as Curve Expert.

Note: This figure is for reference only

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

Three samples of known concentration were tested 20 times on each ELISA plate to assess intraplate precision.

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

Three samples of known concentration were tested 40 times on three different ELISA plates to evaluate the precision of the assay.

Recovery experiments were performed by adding rat a2M at known concentrations to different samples to obtain the recovery range and average recovery.

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

6. The chromogen TMB should be protected from direct sunlight during storage and use. After adding the substrate, carefully observe the color changes in the reaction wells. If a gradient is already evident, terminate the reaction early to avoid excessive color changes that may affect the microplate reader reading.

7. All test tubes and reagents used in the experiment are disposable. Reuse is strictly prohibited, as this will affect the experimental results.

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

9. Components from different batches of the kit should not be mixed (except for the wash solution and the reaction stop solution).

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