I. Sample Processing and Requirements:
1. Serum: Collect whole blood in a serum separator tube and place it at room temperature for 2 hours or at 2-8°C overnight. Then centrifuge at 1000×g for 20 minutes and remove the supernatant. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing.
2. Plasma: Collect the sample using EDTA or heparin as an anticoagulant. Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant and test it. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing.
3. Tissue Homogenate: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove any residual blood (lysed red blood cells in the homogenate may affect the test results). Weigh the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice or in a homogenizer. To further lyse tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes and remove the supernatant for analysis.
4. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, but avoid repeated freeze-thaw cycles.
5. Other biological samples: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis.
6. Sample appearance: The sample should be clear and transparent, and suspended matter should be removed by centrifugation. 7. Sample Storage: Samples collected for testing within one week can be stored at 4°C. If testing cannot be performed promptly, aliquot the sample into single-use portions and store at -20°C (for testing within one month) or -80°C (for testing within six months). Avoid repeated freeze-thaw cycles. Hemolysis of the sample can affect the final test results, so hemolyzed samples are not suitable for this test. II. Required Instruments: 1. Microplate reader (450nm) 2. High-precision pipettes and tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water III. Preparations for the Test: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Preparation of Gradient Standard Working Solution: Add 1 mL of Universal Diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 1000 ng/mL). Then dilute to the following concentrations: 1000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.62 ng/mL, and 0 ng/mL. Serial Dilution Method: Take 7 EP tubes and add 500 μL of Universal Diluent to each tube. Pipette 500 μL of the 1000 ng/mL standard working solution into the first EP tube and mix to make a 500 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details.

3. Preparation of biotinylated antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10uL concentrate + 990uL 4. Preparation of enzyme conjugate working solution: 15 minutes before use, centrifuge 100 μL of concentrated enzyme conjugate at 1000 × g for 1 minute. Dilute 100 μL of concentrated HRP enzyme conjugate with universal diluent to a 1 × working concentration (e.g., 10 μL concentrate + 990 μL universal diluent) and use on the same day. 5. Preparation of 1× Wash Buffer: Dissolve 10 mL of 20× Wash Buffer in 190 mL of distilled water. (Concentrated Wash Buffer removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing the solution.) IV. Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Sample Addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of Universal Diluent to the blank wells. Cover with sealant and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate for testing. This will minimize the impact of matrix effects on the test results. The sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run replicates for all samples and standards.) 3. Adding Biotinylated Antibody: Remove the ELISA plate and discard the liquid. Do not wash. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film sealer and incubate at 37°C for 60 minutes. 4. Washing: Discard the liquid. Add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute. Shake off the wash solution and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Adding Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film sealer and incubate at 37°C for 30 minutes. 6. Washing: Discard the liquid and wash the plate five times as in step 4. 7. Adding substrate: Add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C in the dark for 15 minutes. 8. Adding stop solution: Remove the plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm. V. Calculation of experimental results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction factor. Plot a four-parameter logistic function standard curve on double-logarithmic graph paper with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor. Typical data and reference curves: The following data and curves are for reference only. Experimenters need to establish standard curves based on their own experiments.

Note : This graph is for reference only. The sample content should be calculated based on the standard curve drawn using the same test standard.

VII. Kit Performance:
1. Repeatability: The intra-plate coefficient of variation is less than 10%, and the inter-plate coefficient of variation is less than 10%.
2. Recovery rate: Three different concentration levels of mouse C3 were added to the selected healthy mouse serum and plasma, and the recovery rate was calculated.