Human (cANCA) anti-neutral cytoplasm antibody detection by enzyme-linked immunosorbent assay 1. Remove the kit and allow to stand for 30 minutes at room temperature (20-25°C). 2. Grouping: Remove 96-well plates, determine the number of plates required based on the number of samples to be tested plus the number of standards, and continue to refrigerate the remaining plates. Separately set the standard group (6 concentrations), blank wells, and sample groups to be tested. 3. Dilution of standard products: prepare 6 small test tubes, number them sequentially, add 100 ul of standard diluent into each small test tube, and then add 100 ul of original concentration standard to a well-prepared test tube. Mix well; then take 100ul in the tube and add it to the second tube and mix thoroughly; then add 100ul to the third tube in the tube and mix thoroughly; then take 100ul in this tube and add the fourth one In a test tube, mix thoroughly; take 100 ul of this test tube and add it to the fifth test tube and mix thoroughly; then take 100 ul in the test tube and discard it. The sixth tube was used as standard 0. Note: To reduce experimental errors and ensure accuracy, it is recommended to set up duplicate holes. 4. Add samples: add 50ul of the standard solution to the blank wells according to the order of the standard products; add 50ul of distilled water to the blank control wells; add 40ul to the remaining wells and add 10ul of biotin-labeled antibody. Add the sample to the bottom of the wells of the microtiter plate, try not to touch the wall of the well, mix gently by shaking. 5. Incubation: Seal the plate with a cover plate and incubate at 37°C for 30 minutes. 6. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill the well with washing liquid, and let it stand for 30 seconds. Repeat this 5 times and pat dry. 7. Add enzyme-labeled solution: Add 50 ul of enzyme-labeled solution (excluding blank control wells) to each well of the standard product group and the test sample group. 8. Incubation: The ELISA plate was sealed with a sealing paper and incubated in a humid chamber at 37°C for 1 hour. 9. Wash the plate: Fill each well with the diluted washing solution, and let stand for 15-30 seconds. Thoroughly wash the plate of the enzyme for 5 times. Thoroughly pat dry with absorbent paper. 10. Color development: Add 50 μl of Color A Solution to each well, then add 50 μl of Color B Solution. 11. Stop: React for 10-15 minutes at 25-37°C in the dark and add 50ul stop solution. 12. Read plate: Read the OD value of each well at a wavelength of 450 nm. Note: When you read the plate, you must wipe the traces of liquid and fingers left on the bottom of the plate. The time to read the plate is controlled within 30 minutes after the termination of the reaction so as not to affect the accuracy.