Mouse C4a ELISA Kit

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.

Sample Dilution Guidelines

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

1. 50-fold dilution: One-step dilution. 1. Add 5 μL of sample to 245 μL of Standard & Sample Diluent for a 50-fold dilution.

2. 100-fold dilution: One-step dilution. 1. Add 5 μL of sample to 495 μL of Standard & Sample Diluent for a 100-fold dilution.

3. 1000-fold dilution: Two-step dilution. 1. Add 5 μL of sample to 95 μL of Standard & Sample Diluent for a 20-fold dilution. 2. Add 5 μL of the 20-fold diluted sample to 245 μL of Standard & 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 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 for the sample to be tested through preliminary experiments.

Preparation for Testing

1. 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 any unused solution 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: 10 μg/mL). Subsequently, serially dilute the standard to 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.63 μg/mL, 0.32 μg/mL, and 0.16 μg/mL. Use 0 μg/mL of Standard Diluent as a blank well. Prepare the required amount of standard and set aside. It is recommended to add the prepared standard to the sample within 15 minutes; it is not recommended to leave it 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.

Precautions

1. Before using the kit, ensure that all components are dissolved and mixed thoroughly. Discard any unused standard after reconstitution.

2. Concentrated biotinylated antibody and enzyme conjugate are small in size and may disperse throughout the tube during transportation. Before use, centrifuge at 1000×g for 1 minute to allow any liquid on the tube walls or cap to settle to the bottom. Mix the solution by carefully pipetting 4-5 times before use. Prepare the standard, biotinylated antibody working solution, and enzyme conjugate working solution according to the required volume and use the corresponding diluents. Do not mix them.

3. Concentrated wash buffer may crystallize after removal from the refrigerator. This is normal. Dissolve the crystals completely in a water bath or incubator before preparing the wash buffer (do not heat above 40°C). The wash buffer should be at room temperature when used.

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 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 from different batches cannot be mixed (except washing solution and reaction stop solution).

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

Operation Steps

1. Before starting the experiment, all reagents should be equilibrated to room temperature and all reagents should be prepared in advance. When diluting reagents or samples, they must be mixed thoroughly, and try to avoid foaming during mixing. If the sample concentration is too high, dilute it with sample diluent so that the sample is 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 well walls. 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 experimental 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. The instrument should be preheated and the assay program set before use. Calculation of Results: 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. 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 plotting. At the same time, to ensure intuitive experimental results, the graph provides raw data rather than logarithmic values. Due to differences in experimental operating conditions (such as operator, pipetting technique, plate washing technique, and temperature conditions), the OD values of the standard curve will vary. The standard curve provided is for reference only; experimenters should establish a standard curve based on their own experiments. The OD value of the used sample can be used to calculate the sample concentration on the standard curve. Multiplying by the dilution factor will give the actual sample concentration. It is recommended to use professional curve drawing software, such as Curve Expert.

Note: This image is for reference only.

• Precision

Intra-plate precision (within-assay precision): CV%<8%

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

Inter-assay precision (between-assay precision): CV%<10%

Three samples of known concentrations were each tested 40 times on three different ELISA plates to assess inter-assay precision.

• Recovery Rate

Recovery rate experiments were performed by adding known concentrations of mouse C4a to different samples to obtain the recovery rate range and average recovery rate.

• Linearity

The samples spiked with mouse C4a were diluted 2-fold, 4-fold, 8-fold, and 16-fold for recovery experiments, and the recovery rate range was obtained.

Chinese name

96T

Save conditions

ELISA plate (removable)

12 strips x 8 Well

4°C/-20°C

Lyophilized Standard

2

4°C/-20°C

Standard & Sample Dilution

20 mL

Concentrated Biotinylated Antibody (100×)

120 μL

4°C/-20°C

Biotinylated Antibody Dilution Buffer

12 mL

4°C/-20°C

Concentrated HRP enzyme conjugate (100×)

120 μL

4°C/-20°C

Enzyme Conjugate Dilution Buffer

12 mL

4°C/-20°C

Concentrated Wash Buffer (25×)

20 mL

4°C/-20°C

Chromogenic substrate solution (TMB)

10 mL

4°C/-20°C (protect from light)

Reaction stop solution

6 mL

4°C/-20°C

Sealing film

2

Normal temperature