| Usage | Sample Processing and Requirements: Tissue Homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and mince the tissue. 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. 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 collect the supernatant for analysis. Cell Lysis Solution: Gently wash adherent cells with pre-chilled PBS, then trypsinize and harvest the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Wash the collected cells three times with pre-chilled PBS and resuspend in 150-200 μL of PBS per 1 × 10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, reduce the PBS volume appropriately). Disrupt the cells by repeated freeze-thaw cycles or sonication. Centrifuge the extract at 1500 × g for 10 minutes at 2-8°C, and remove the supernatant for analysis.
Cell culture supernatant: Centrifuge at 1000 × g for 20 minutes, remove the supernatant, or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles.
Other biological fluids: Centrifuge at 1000 × g for 20 minutes, remove the supernatant, and analyze.
Pre-assay preparation:
1. Remove the reagent kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a 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 pg/mL). Then dilute to the following concentrations: 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.625 pg/mL, and 0 pg/mL. Serial dilution method: Add 500 μL of universal diluent to each of seven EP tubes. Pipette 500 μL of the 1000 pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500 pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is directly used as a blank well, and there is no need to aspirate liquid from the penultimate tube.
3. Preparation of biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use, and dilute the 100× concentrated biotinylated antibody to 1× working concentration with universal diluent (e.g. 10uL concentrate + 990uL universal diluent), and use it immediately. 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 the 100 μL concentrated HRP conjugate with universal diluent to a 1 μL working concentration (e.g., 10 μL concentrate + 990 μL universal diluent). Prepare freshly for use. 5. Preparation of 1 μL Wash Solution: Dissolve 10 mL of 20 μL Wash Solution in 190 mL of distilled water. (Concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing.) 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 a film sealer 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 plate. This will minimize matrix effects on the test results. The sample concentration should be multiplied by the dilution factor when calculating the final sample concentration. It is recommended to run replicates for all samples and standards.) 3. Biotinylated Antibody Addition: Remove the 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. Wash: Discard the liquid and add 300 μL of 1x wash buffer to each well. Let stand for 1 minute. Shake off the wash buffer and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add 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. Wash: Discard the solution and wash the plate five times according to the washing method in step 4. 7. Add Substrate: Add 90 μL of substrate (TMB) to each well, cover with a film sealer, and incubate at 37°C in the dark for 15 minutes. 8. Add 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 450 nm. 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 value. Plot a standard curve for the four-parameter logistic function 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 and retest. Multiply the sample concentration by the corresponding dilution factor. |
| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with Interleukin 4 (IL4) capture antibody. After incubation and washing, the assay is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of Interleukin 4 (IL4) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. |
| Source | Rabbit |
| Synonym | Rabbit Interleukin 4 ELISA Kit |
| Detection Type | Double antibody sandwich method |
| Composition | | Name | 9 6 T Configuration | Remarks | | Pre-coated 96-well enzyme plate | 8 holes×12 strips | None | | Standard | 2 bottles | Dilution according to the instructions | Universal diluent | 2×20mL | None | Concentrated biotinylated detection antibody (100×) | 120uL | Concentrated enzyme conjugate (100×) | 120uL | Dilution according to the instructions | 20× Wash solution | 2×10mL | Dilute according to instructions | Substrate (TMB) | 10mL | None | Stop solution | 6mL | none | Sealing film | 4 sheets | None | Instructions | 1 serving | None |
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| Background | Interleukin-4 (IL-4) is a multi-effect cytokine secreted primarily by a variety of cells, including basophils, eosinophils, mast cells, and Th2 cells, and exerts immunoregulatory functions in many cells. As an activating factor for B cells, IL-4 regulates the type switching of immunoglobulins on the B cell surface, particularly between IgG and IgE. IL-4 also regulates the differentiation of precursor helper T cells into the Th2 subtype. IL-4, in synergistic action with TNF-α, can induce the expression of VCAM-1 on the surface of vascular endothelial cells and smooth muscle cells, thereby selectively recruiting eosinophils and lymphocytes to sites of inflammation. |
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use.
2. Improper plate washing may lead to inaccurate results. Ensure that the wells are completely aspirated before adding substrate. Do not allow the wells to dry out during incubation.
3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value.
4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue.
5. Avoid cross-contamination of reagents and specimens to prevent erroneous results.
6. Avoid direct exposure to strong light during storage and incubation.
7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit.
8. Do not use expired products, and do not mix components from different product numbers and batches. 9. Recombinant proteins from sources other than the test kit may not match the antibodies in this kit and may not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and the samples and detection devices should be handled according to the prescribed procedures. |
| Storage Temp. | Store at 2-8°C. Valid for 6 months. |
| Test Range | 15.6-1000 pg/mL |
| Applications | Tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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