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Operational process

• Coating antibody: Dilute the coating antibody to the required dilution using carbonate buffer (CBS) or phosphate-buffered saline (PBS) according to the experimental needs. Coat 100 μL per well, incubate at 37°C for 2 hours or at 4°C overnight.
• Discard the liquid in the wells, dry, wash the plate twice with 10 mM PBST (10 mM PBS + 0.05% Tween-20), each time soaking for 1-2 minutes, 350 μL per well, and dry (you can also gently tap to remove the liquid from the wells).
• Block with 10 mM PBST (10 mM PBS + 0.05% Tween-20) containing either 1% BSA or 5% skim milk, using 350-400 μL per well, at 37°C for 2 hours.
• Wash the plate: follow step 2.                Note: Commercial kits usually come pre-coated with capture antibodies on the ELISA plates, so steps 1-4 are generally unnecessary. Please verify before use.
• Add samples: Set up zero wells, standard wells, and sample wells. Add 100 μL of sample diluent to the blank wells, and add 100 μL of standard or sample to the remaining wells                Note: avoid bubbles, add the sample to the bottom of the well, try not to touch the well wall, and complete the addition of samples on one plate within 10 minutes.                Cover the enzyme-linked immunosorbent assay (ELISA) plate with a lid or film, and incubate at 37°C for 60-120 minutes. To ensure the validity of the experimental results, please use a new standard solution for each experiment.
• Discard the liquid in the wells, dry, wash the plate 4 times with 10 mM PBST (10 mM PBS + 0.05% Tween-20), each time soaking for 1-2 minutes, using 350-400 μL per well, and dry (you can also gently tap to remove the liquid from the wells).
• Add detection antibody: According to the experimental requirements, dilute the detection antibody with PBST to a certain dilution, 100 μL per well, at 37°C for 1 hour.
• Wash the plate: as step 6.
• Add secondary antibody: Dilute the secondary antibody with 10 mM PBST to the required dilution, add 100 μL per well, incubate at 37°C for 1 hour.
• Wash the plate: as step 6.
• Add TMB: Add 100μL of TMB substrate solution to each well, incubate at 37°C in the dark for 10-30 minutes (adjust the time according to the experimental requirements to avoid excessive color development).
• Add stop solution: After the color development reaches the expected level (such as a gradient color appearing in the standard wells), add 50μL of stop solution (e.g., 2M citric acid) to each well to terminate the enzymatic reaction (at which point the blue color changes to yellow).
• Enzyme-linked immunosorbent assay (ELISA) reading: Using 630 nm as the calibration wavelength, measure the absorbance (OD value) of each well at 450 nm with an ELISA reader in sequence. Read the values within 5 minutes after adding the stop solution.