Washing method for human connective tissue activating peptide III (CTAP III) ELISA kit:

1. Automatic washing machine: Add 350 μ l of washing solution to each well, with a 60 second interval between injection and suction. Wash the board 5 times.

2. Manual plate washing: Shake off the liquid in the holes and pat dry on clean absorbent paper. Add 350 μ l of washing solution to each hole and soak for 1-2 minutes. Remove the liquid from the enzyme-linked immunosorbent assay plate (not touching the plate wall) or shake off the liquid, and pat dry on thick absorbent paper. Wash the board 5 times.

Before the experiment begins, all reagents should be equilibrated to room temperature; When preparing reagents or samples, they should be thoroughly mixed and foaming should be avoided as much as possible.

1. Sample addition: Set up blank holes, standard holes, and sample holes for testing. Add 100 μ l of sample diluent to the blank well, and add 100 μ l of standard or test sample to the remaining wells respectively. Be careful not to have bubbles. When adding the sample, place it at the bottom of the enzyme-linked immunosorbent assay plate, try not to touch the well wall, and gently shake and mix well. Cover the enzyme-linked immunosorbent assay (ELISA) plate with film and incubate at 37 ℃ for 2 hours. To ensure the validity of the experimental results, please use a new standard solution for each experiment.

2. Discard the liquid, shake dry, and add 100 μ l of Detection Ab working solution (prepared within 15 minutes before use) to each well. Cover the enzyme-linked immunosorbent assay plate with a film and incubate at 37 ℃ for 1 hour.

3. Discard the liquid in the hole, shake dry, wash the plate 3 times, soak for 1-2 minutes each time, about 350 μ l/hole, shake and lightly tap on absorbent paper to dry the liquid in the hole.

Dry 4. Add 100 μ l of HRP Conjugate working solution (prepared within 15 minutes before use) to each well, cover with film, and incubate at 37 ℃ for 1 hour.

5. Discard the liquid in the hole, shake dry, and wash the plate 5 times using the same method as step 3.

6. Add 100 μ l of substrate solution to each well, and incubate the enzyme-linked immunosorbent assay (ELISA) plate with a film at 37 ℃ in the dark for about 15 minutes (shorten or prolong according to the actual color development situation, but not exceed 30 minutes. When a significant gradient appears in the standard well, it can be terminated).

7. Add 50 μ l of termination solution to each well to terminate the reaction, at which point the blue color will turn yellow. The order of adding termination solution should be as similar as possible to the order of adding substrate solution.

8. Immediately measure the optical density (OD value) of each well using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450nm. The power of the enzyme-linked immunosorbent assay (ELISA) reader should be turned on in advance, the instrument should be preheated, and the detection program should be set up.

After the experiment is completed, the unused reagents should be stored in the refrigerator at the specified temperature until the expiration date.