(1) Principle ELISA is based on immunological reaction, a highly sensitive test technique that combines the specific reactions of antigens and tethers with the efficient catalytic effect of enzymes on substrates. Since the reaction of antigen and antibody is carried out in the well of a solid phase carrier-polystyrene microtiter plate, after each reagent is added to the incubation, excess free reactants can be removed by washing to ensure the specificity of the test results With stability. In practical applications, through different designs, there are many specific method steps. That is: the indirect method for detecting antibodies (Figure a), the double antibody sandwich method for detecting antigen (Figure b), and the antigen competition method for detecting small molecule antigens or haptens, etc. More commonly used are ELISA double antibody sandwich method and ELISA indirect method. (B) Operation steps Method 1 Double antibody sandwich method for detecting unknown antigens: (3) Reagent equipment 1. Reagents (1) Coating buffer (PH9.6 0.05M carbonate buffer): (4) Matters needing attention 1. During 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 high, indicating a non-specific reaction, which can be blocked with sheep serum, rabbit serum or BSA.
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ELISA is the abbreviation for Enzyme-Linked Immunosorbnent Assay. It is an immunoenzyme technology developed after immunofluorescence and radioimmunoassay. This technology has developed very rapidly since its introduction in the early 1970s, and it has been widely used in many fields of biology and medical sciences.
1. Coating: Dilute the antibody with 0.05M PH9. Carbonate coating buffer to a protein content of 1 ~ 10μg / ml. 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. Sample addition: 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. O · D value can also be measured: On an ELISA detector, at 450nm (if ABTS color is developed, then 410nm), the blank control well is zeroed and the O · D value of each well is measured. 2.1 times the value is positive.
Method 2: Indirect method for detecting unknown antibodies:
Dilute the known antigen to 1 ～ 10μg / ml with coating buffer, add 0.1ml per well, and overnight at 4 ℃. Wash 3 times the next day. Add a certain dilution of the test sample (unknown antibody) 0.1ml to the above-mentioned coated wells, incubate at 37 ° C for 1 hour, and wash. (Make blank, negative and positive well control at the same time) Add 0.1ml fresh diluted enzyme-labeled secondary antibody (anti-antibody) to the reaction well, incubate at 37 ℃ for 30-60 minutes, wash, and finally wash with DDW. The remaining steps are the same as 4, 5 and 6 of the "double antibody sandwich method".
NaCO3 1.59g NaHCO3 2.93g Add distilled water to 1000ml
(2) Wash buffer (PH7.4 PBS): 0.15M KH2PO4 0.2 g Na2HPO4 · 12H2O 2.9 g NaCl 8.0 g KCl 0.2 g Tween-20 0.05% 0.5ml Add distilled water to 1000ml
(3) Diluent: bovine serum albumin (BSA) 0.1g, add washing buffer to 100ml, or use sheep serum, rabbit serum and other serum to mix with the washing solution to 5-10%.
(4) Stop solution (2M H2SO4): 178.3ml of distilled water, 21.7ml of concentrated sulfuric acid (98%) was added dropwise.
(5) Substrate buffer (PH5.0 phosphate date citric acid): 0.2M Na2HPO4 (28.4g / L) 25.7ml 0.1M citric acid (19.2g / L) 24.3ml plus 50ml of distilled water.
(6) TMB (tetramethylbenzidine) using solution: TMB (10mg / 5ml absolute ethanol) 0.5ml substrate buffer solution (PH5.5) 10ml 0.75% H2O2 32μl
(7) ABTS solution: ABTS 0.5mg substrate buffer (PH5.5) 1ml 3% H2O2 2μl
(8) Antigen, antibody and enzyme-labeled antibody.
(9) Normal human serum and positive control serum.
2. Equipment:
(1) 40- or 96-well polystyrene plastic plate (abbreviated as enzyme-labeled plate), 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.
2. In ELISA, the selection of various experimental conditions is very important, including:
(1) Selection of solid-phase carrier: many substances can be used as solid-phase carrier, such as polyvinyl chloride, polystyrene, polyacrylamide and cellulose. The form can be concave hole plate, test tube, bead, etc. At present, the commonly used 40-hole polystyrene concave orifice plate. No matter what kind of carrier, it can be screened before use: it is coated with the same amount of antigen, and the reaction is carried out under the same experimental conditions. Observe whether the color reaction is uniform and determine whether its adsorption performance is good.
(2) The choice of coating 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 during adsorption. Adsorption temperature, time and protein content also have a certain influence, generally use 4 ℃ for 18-24 hours. The optimal concentration of protein coating needs to be titrated: that is, after coating with different protein concentrations (0.1, 1.0, and 10 μg / ml, etc.), when the other test conditions are the same, observe the OD value of the positive specimen. Choose the concentration with the largest OD and the least protein. For most proteins, it is usually 1 ～ 10μg / ml.
(3) Selection of working concentration of enzyme-labeled antibody: firstly, the initial titer is titrated by direct ELISA method (see enzyme-labeled antibody section). Then fix other conditions or adopt the "square matrix method" (the coating, the reference of the sample to be tested and the enzyme-labeled antibody are different dilutions) to accurately titrate its working concentration in the formal experimental system.
(4) Selection of enzyme substrates and hydrogen donors: The selection requirements for hydrogen donors are inexpensive, safe, and have a significant color reaction, but are colorless in themselves. Some hydrogen donors (such as OPD, etc.) have potential carcinogenic effects and should be protected. Those with conditions should use non-carcinogenic, highly sensitive hydrogen donors, such as TMB and ABTS, which are currently more satisfactory hydrogen donors. After the substrate acts for a period of time, a strong acid or base should be added to stop the reaction. Usually the substrate action time is 10-30 minutes. Substrate use solution must be freshly prepared, especially H2O2 added before use
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