1. In the formal test, the test conditions should be controlled by the positive control and the negative control respectively, and the samples to be tested should be made in duplicate to ensure the accuracy of the experimental results. Sometimes the background is higher, indicating a non-specific reaction, which can be blocked by sheep serum, rabbit serum or BSA. 2. In the ELISA , it is important to select the various experimental conditions, including: (1) Selection of solid phase support: Many substances can be used as solid phase carriers such as polyvinyl chloride, polystyrene, polyacrylamide and cellulose. The form may be a flat plate, a test tube, a bead or the like. Currently used is a 40-well polystyrene recessed plate. Regardless of the carrier, screening can be carried out before use: the reaction is carried out under the same experimental conditions by coating with an equal amount of antigen, and whether the coloration reaction is uniform or not, and whether the adsorption performance is good. (2) Selection of coated antibody (or antigen): When the antibody (or antigen) is adsorbed on the surface of the solid phase carrier, the purity is required to be good, and the pH is generally required to be between 9.0 and 9.6. The adsorption temperature, time and the amount of protein also have a certain influence, and generally use 4 ° C for 18 to 24 hours. The optimum concentration of protein coating is titrated: after coating with different protein concentrations (0.1, 1.0, and 10 μg/ml, etc.), the OD value of the positive specimen is observed when the other test conditions are the same. The concentration with the highest OD value and the least amount of protein is selected. It is usually 1 to 10 μg/ml for most proteins. (3) Selection of working concentration of enzyme-labeled antibody: First, titration of preliminary titer is carried out by direct ELISA (see enzyme-labeled antibody fraction). Then, other conditions are fixed or the "square matrix method" (the coating, the reference sample of the sample to be tested, and the enzyme-labeled antibody are respectively different dilutions) are accurately titrated in the formal experimental system. (4) Enzyme substrate and hydrogen donor selection: The choice of hydrogen donor is cheap, safe, and has obvious color reaction, but it is colorless. Some hydrogen donors (such as OPD) have potential carcinogenic effects and should be protected. Those who are qualified should use hydrogen donors that are not carcinogenic and sensitive. For example, TMB and ABTS are currently satisfactory hydrogen donors. After the substrate has been applied for a while, a strong acid or a strong base should be added to terminate the reaction. Usually the substrate action time is preferably 10-30 minutes. The substrate used must be freshly prepared, especially H2O2 before use. 3. Specimen collection: The quality of specimen collection and preservation directly determines whether the experiment can be successful. The requirements for specimen collection and storage must be considered before collection of specimens. These requirements have a great impact on the stability of the analytes in the specimens. Take ELISA as an example. The ELISA test is generally a liquid specimen. Immediately after collecting the fresh specimen, centrifuge the supernatant for about 1 ml and transfer it to a high-temperature sterilized 1.5 ml EP tube. This specimen was stored at 4 degrees for 48 hours, -20 degrees for 1 month, and -80 degrees for 6 months. Samples are generally afraid of repeated freezing and thawing, which will cause protein degradation. If you need to do pre-experiment or need to take samples repeatedly, you should pack several EP tubes at the time of collection (note the label with a pen outside the tube) . 4. Preparation before the experiment: 100μl sample gun and pipette tip, 1000μl sample gun and pipette tip, 100ml measuring cylinder (washed and double distilled water), 500ml measuring cup (washed and double distilled water, the laboratory did not Smaller), double distilled water 100ml (diluted washing solution), blown balloon, absorbent paper (qualitative filter paper), test tube rack, dark box, dark plate, ELISA plate, timer, tape, scissors, waste box and a waste container; in 100 l pipette tip, 1000μl large tip, 1.5ml EP tube with prior sterilization good; microplate box drying should be washed repeatedly used. 5. When preparing the experiment, warm the 37 degree incubator to 37 degrees in advance; the sample and kit are equilibrated at room temperature for more than 30 minutes. 6. The antibody immobilized by the ELISA kit is at the bottom. Do not touch the bottom of the gun when loading the sample; use the incomplete kit to separate it, but do not touch the bottom at the same time. The reason is the same as before. 7. Seal the plate opening with tape at each incubation to prevent evaporation of water. 8. The microplates are generally in a row of eight, and the edges of the heads at one end are marked with a marker (such as 1, 2, 3...) to avoid accidental shots when the board is shot down. The microplate fell out of the ELISA plate and the sequence could not be confirmed. 9. Replace the tip with a standard and sample each time to avoid cross-contamination by repeated use of the tip. 10. After 37 degrees of incubation, do not forget to pour off the liquid in the microplate (including the unconjugated enzyme-linked affinities, the residue must cause false positives), and repeatedly wash the plate with diluted detergent. 11. Pre-experiment and standard curve should be done. If most of the sample values ​​fluctuate at the highest OD value, dilute and redo, then calculate the final result according to the dilution factor, because the kit has a test range. Related Technical Services: ELISA Testing Services Tiamulin Fumarate,Tiamulin Swine,Tiamulin Chicken,Tiamulin For Animal Use Shandong Shengli Bioengineering Co., Ltd , https://www.shenglipharm.com
ELISA experiment considerations