PROG ELISA Kit
PROG ELISA Kit
Product Details
Product Details
Product Specification
| Usage | I. Sample Handling and Requirements: 1. The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. Before the experiment, it is recommended to estimate the analyte concentration in the sample based on relevant literature and conduct preliminary experiments to determine the actual concentration. If the analyte concentration in the sample is too high or too low, please dilute or concentrate the sample appropriately. 2. If the sample type to be tested is not listed in the instructions, it is recommended to conduct preliminary experiments to verify the validity of the assay. 3. Serum: Collect whole blood in a serum separator tube and incubate at room temperature for 2 hours or at 2-8°C overnight. Then, centrifuge at 1000×g for 20 minutes. The supernatant can be collected or stored at -20°C or -80°C. Avoid repeated freezing and thawing. 4. Plasma: Collect the sample using EDTA or heparin as an anticoagulant. Centrifuge the sample at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 5. 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 may affect 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., 1 g of tissue sample to 9 mL of PBS. The specific volume can be adjusted based on experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and thoroughly grind on ice or in a homogenizer. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and remove the supernatant for testing. 6. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes, remove the supernatant, and test immediately. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing. 7. Other biological samples: Centrifuge at 1000×g for 20 minutes, remove the supernatant, and test immediately. 8. Sample appearance: The sample should be clear and transparent, and any suspended matter should be removed by centrifugation. 9. Sample storage: Samples collected for testing within one week can be stored at 4°C. If testing is not possible, aliquot the sample into a single-use aliquot and freeze at -20°C (for testing within one month) or -80°C (for testing within six months). Avoid repeated freezing and thawing. Hemolysis of the sample can affect the final test results, so hemolyzed samples are not suitable for this test. 2. Equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipettes and tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C constant temperature box 4. Distilled or deionized water III. Preparation before testing: 1. Please take out the test kit from the refrigerator 10 minutes in advance and equilibrate it 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 5000 pg/mL). Then dilute to the following concentrations: 5000 pg/mL, 2500 pg/mL, 1250 pg/mL, 625 pg/mL, 312.5 pg/mL, 156.25 pg/mL, 78.12 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 5000 pg/mL standard working solution into the first EP tube and mix thoroughly to make a 2500 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 draw liquid from the penultimate tube, as shown in the figure below. 3. Preparation of HRP-antigen working solution: 15 minutes before use, centrifuge the concentrated HRP-antigen at 1000×g for 1 minute. Dilute 100× concentrated HRP-antigen to a 1× working concentration with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent) and use on the same day. 4. 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 film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1x with universal diluent before adding it to the ELISA plate for testing. This will reduce 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 set up duplicate wells for all samples and standards.) 3. Wash: Discard the liquid and add 300uL of 1x wash buffer to each well. Let it stand for 1 minute. Shake off the wash buffer and pat dry on absorbent paper. Repeat this process three times (a microplate washer can also be used). 4. Add substrate: Add 90uL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 minutes. 5. Add stop solution: Remove the ELISA plate and add 50uL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450nm. V. Calculation of Experimental Results: 1. Calculate the average OD value of the replicate wells containing the standard and sample and subtract the OD value of the blank well as the correction value. 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 when calculating the sample concentration. Typical Data and Reference Curves: The following data and curves are for reference only. Experimenters should establish a standard curve based on their own experiments.
6. Kit performance: |
