| Usage | Experimental equipment required for the experiment:
1. Microplate reader (450nm)
2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL
3. 37℃ constant temperature box
4. Distilled water or deionized water
Sample processing and requirements:
Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen 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. 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 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 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 frozen and thawed. Finally, centrifuge the homogenate at 5000 × g for 5-10 minutes, and the supernatant can be assayed. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect them by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with ice-cold PBS and resuspend them 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, but avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge at 1000×g for 20 minutes, and remove the supernatant for analysis. 1. First, bring the kit to room temperature (approximately 30 minutes), then prepare the wash buffer to the working concentration with purified water (one part concentrated wash buffer to 19 parts purified water). 2. Remove the ELISA plate. Each specimen requires 8 wells, 8 wells for the positive control, and 8 wells for the negative control. Store any excess in a ziplock bag, remembering to include desiccant. 3. Add 50 μl of specimen diluent to each well of the specimen test well. Then, add 50 μl of cell culture supernatant (or affinity-purified antibody) to the ELISA microplate, adding 8 wells per specimen and 100 μl per well for each of the positive and negative controls. No specimen diluent was added. Apply sealing film and incubate at 37°C for 30 minutes. 4. Discard the liquid from the plate, wash the plate 5 times, and then pat dry on a non-linting absorbent material or machine wash 5 times. Then, add 100 μl of each of the eight enzyme-labeled secondary antibodies to each of the eight wells containing each specimen, and the same applies to the eight wells containing the positive and negative controls. Apply a sealing film and incubate at 37°C for 30 minutes. 5. Aspirate the plate, wash the plate five times, and then pat dry on a non-linting absorbent material or machine wash five times. Add 50 μl each of chromogen A and chromogen B to each well. Replace the sealing film, apply the plate, and develop the color at 37°C in the dark for 20 minutes. 6. This kit has high specificity; the results can generally be observed visually. The enzyme-labeled secondary antibody corresponding to the well that displays blue color will indicate the Ig class or subclass of the specimen. You can also stop the reaction with the stop solution (50ul per well) and then use a microplate reader to measure the results at a dual wavelength of 450nm and 630nm as a reference wavelength. By referring to the enzyme-labeled secondary antibody corresponding to the high-value wells, you can know the Ig class or subclass of the specimen (the positive control OD is generally not less than 0.8, and the negative control is generally not higher than 0.12. The positive judgment standard is: the sample OD is greater than the negative OD + 0.15, and the negative OD is lower than 0.05, which is calculated as 0.05). |
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