As another example, when changing from a lying position to a standing position, the serum renin activity will increase significantly. Another example is the testing of therapeutic drugs, which should be selected according to the pharmacokinetics of the most appropriate time to take blood test. The collection of serum samples used for the detection of antigens and antibodies, tumor markers and special proteins of infectious pathogens has no effect on time and body position. Operation steps of ELISA kit: method one: double antibody sandwich method for detecting unknown antigens 1. Coating: Dilute the antibody to protein content of 1-10 μg / ml with 0.05MPH9. Carbonate coating buffer. Add 0.1ml to the reaction well of each polystyrene plate, overnight at 4 ℃. The next day, the solution in the well was discarded and washed 3 times with washing buffer for 3 minutes each time. (Referred to as washing, the same below). 2. Add sample: add 0.1ml of the diluted test sample to the above-mentioned coated wells and incubate at 37 ℃ for 1 hour. Then wash. (Make blank wells, negative control wells and positive control wells at the same time). 3. Add enzyme-labeled antibody: add 0.1ml of freshly diluted enzyme-labeled antibody (dilution after titration) to each reaction well. Incubate at 37 ° C for 0.5 to 1 hour and wash. 4. Add substrate liquid to develop color: add 0.1ml of temporarily prepared TMB substrate solution to each reaction well at 37 ℃ for 10-30 minutes. 5. Terminate the reaction: Add 0.05ml of 2M sulfuric acid to each reaction well. 6. Judgment of results: The results can be directly observed with the naked eye on a white background: the darker the color in the reaction well, the stronger the positive degree, and the negative reaction is colorless or extremely light. "-" Indicates. OD value can also be measured: on the ELISA detector, at 450nm (410nm if ABTS color is developed), the OD value of each well is measured after zero adjustment with a blank control well, if it is greater than 2.1 times the specified negative control OD value , That is positive. Method 2: Indirect method for detecting unknown antibodies 1. Dilute the known antigen to 1 ～ 10μg / ml in coating buffer, add 0.1ml per well, and overnight at 4 ℃; 2. Wash 3 times the next day; 3. Add Dilute 0.1ml of the test sample (unknown antibody) in the above-mentioned coated wells, incubate at 37 ° C for 1 hour, and wash; 4. (Also do blank, negative and positive well controls) in the wells, add 0.1ml freshly diluted enzyme-labeled secondary antibody (anti-antibody); 5. Incubate for 30-60 minutes at 37 ° C, wash; 6. Wash with DDW on the last pass. The remaining steps are the same as 4, 5 and 6 of the "double antibody sandwich method". Reagent (1) Coating buffer (PH9.60.05M carbonate buffer): Na2CO3 1.59g NaHCO3 2.93g Add distilled water to 1000ml (2) Wash buffer (PH7.4PBS): 0.15M KH2PO4 0.2g Na2HPO4 · 12H2O 2.9g NaCl 8.0g KCl 0.2g Tween-20 0.05% 0.5ml add distilled water to 1000ml (3) Diluent: bovine serum albumin (BSA) 0.1g plus wash buffer to 100ml or use sheep serum, rabbit serum and Other serum and Use 5-10% of the washing solution. (4) Stop solution (2M H2SO4): 178.3ml of distilled water, 21.7ml of concentrated sulfuric acid (98%) is added dropwise. (5) Substrate buffer solution (PH5.0 phosphate date citric acid): 0.2MNa2HPO4 (28.4g / L) 25.7ml 0.1M citric acid (19.2g / L) 24.3ml Add 50ml of distilled water. (6) TMB (tetramethylbenzidine) use solution: TMB (10mg / 5ml absolute ethanol) 0.5ml substrate buffer solution (PH5.5) 10ml 0.75% H2O2 32μl (7) ABTS use solution: ABTS 0.5mg base Buffer (PH5.5) 1ml 3% H2O2 2μl (8) Antigen, antibody and enzyme labeled antibody. (9) Normal human serum and positive control serum. Equipment (1) Polystyrene plastic plate (abbreviated as enzyme-labeled plate) 40-well or 96-well, ELISA detector, 50μl and 100μl sampler, plastic dripper, small towel, washing bottle. (2) Small beakers, glass rods, test tubes, straws and measuring cylinders. (3) 4 ℃ refrigerator, 37 ℃ incubator.
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