IVD Research Dog teaches you how to optimize ELISA!
1. Selection of solid phase carrier
There are many types of carriers, including cellulose, cross-linked dextran, staphylococcal polystyrene, polyacrylamide, etc. The forms of use include concave plates, test tubes, beads, etc.
Polystyrene concave orifice plate is the most widely used carrier. Polystyrene plastic microtiter plates have good protein adsorption performance, are easy to operate, use small amounts, and are suitable for large-scale inspections.
Because the polystyrene process is not stable enough, the differences between batches are large. Therefore, screening must be performed before performing ELISA. Inspection Method:
⑴ Check the adsorption performance: first add antibody coating, then add enzyme-labeled antibody of the same dilution, finally add substrate, develop color, measure OD value, calculate the overall average OD value, and then calculate the OD value of each two adjacent wells. OD average value, this average value is qualified within ±10% of the total average value. If the OD value of the middle hole and the surrounding holes is too different, or the OD value of one side of the hole is different from the other side hole, it is considered unqualified.
⑵ Compare and measure the positive serum and negative serum to observe whether there is any obvious difference. If the OD value of the two differs by more than 10 times, it is qualified.
Board handling. New boards generally do not need to be treated and can be used after being rinsed with distilled water. The board is discarded after one use. However, many experimental workers believe that ultrasonic treatment and cleaning solution Tritonx100 and 20% ethylene glycol can still be used. However, if it is found that the blank control has a darker color and the positive sample has unsatisfactory color results, it should be discarded.
2. Adsorption conditions of the carrier
The adsorption of carriers is all physical adsorption. The amount of adsorption depends on pH value, temperature, protein concentration, ionic strength and adsorption time.
Better adsorption conditions are: ionic strength is 0.05Mol/L~0.10Mol/L, pH9.0~pH9.6 carbonate buffer, protein concentration is 1μg/ml~100μg/ml, 4℃ overnight or 37℃ 3h.
3. Determination of the concentration of enzyme-labeled antibodies used
Add a sufficient amount of antibody to the wells of the polystyrene plate to coat, incubate for a certain period of time, rinse, and dilute the enzyme-labeled conjugate. Add 2 wells for each dilution, incubate, rinse, and add substrate to develop color. ,Colorimetric. Use the OD value as the ordinate and the dilution of the enzyme-labeled conjugate as the abscissa to create a curve. Find the optimal enzyme-labeled antibody dilution when the OD value is 1.
This dilution refers to the optimal dilution under this condition. It would not be optimal under other conditions. For example, incubation of an enzyme-labeled antibody at a dilution of 1:400 for 6 hours can produce the same results as incubation at a dilution of 1:6,400 for 24 hours. Therefore, once the conditions are determined, do not change them to ensure the repeatability and relative accuracy of the results.
This optimal enzyme-labeled antibody dilution can be used as the working concentration, or it can be increased by half to one titer, but it cannot be increased too high, otherwise non-specific color development will increase.
The titer of the enzyme-labeled antibody reflects the quality of the enzyme-labeled antibody, and can also be used to compare the quality of the enzyme-labeled conjugates. Some reports believe that a titer of 1:320 is qualified, and a titer of 1:1,000 or above is better. The higher the titer of the enzyme-labeled antibody, the greater the dilution factor used for the working concentration, the higher the sensitivity, and the lower the non-specific reaction.
4. Antigen:
⑴ Antigen requirements:
The antigen used for ELISA must be fairly pure. If it contains other impurities, it will compete with the antigen for limited positions on the solid-phase carrier. Antigens used for other serological reactions may not be suitable for ELISA experiments and must be tested. The antigen must be firmly adsorbed on the carrier without losing its immune activity, and regular and repeated results can be obtained. In addition, after adsorbing the carrier, there is minimal non-specific adsorption of various added reagents, that is, there is a large difference in binding with negative and positive serum.
⑵ Determination of antigen titer:
Single square array or double array testing can be used. ①Single array test: Coat the enzyme plate with antigens of different dilutions, add the conventional 1:200-fold diluted positive serum, then add the enzyme-labeled antibody, develop color, and measure the OD value. When the OD value is 1.0 , the corresponding antigen concentration is used as the titer; ② The double-array test is more accurate, and it can measure the optimal concentration of both the antigen and the antibody. That is, the antigen and antibody are diluted into different concentrations for enzyme-labeled antibody reaction. The dilution of the antigen corresponding to the largest difference in light absorption value between the negative and positive serum with the highest dilution multiple is the titer of the antigen.
The solid-phase carrier of the adsorbed antigen is very stable after freeze-drying or drying and will not become inactive for several months.
5. Cleaning fluid
Generally, 0.01Mol/L pH 7.2 PBS Tween buffer is used. Tween is polyoxyethylene sorbitan fatty acid ester, which is a non-ionic surface tension substance and is often used as a co-solvent. The number of Tween depends on the type of fatty acid to which the polymerized sorbitol is combined. Tween 20 is combined with lauric acid, Tween 40 is combined with palmitic acid, Tween 60 is combined with stearic acid, Tween 80 is combined with oleic acid, etc. Tween 20 is usually added to the buffer as a wetting agent to reduce non-specific adsorption. You can also add 1% bovine serum albumin (or 10% calf serum or ovalbumin) to the PBS buffer, especially after the antigen is coated, coat it again with bovine serum albumin buffer to occupy the wells. Leave the remaining positions to reduce non-specific reactions.
6． Reaction time
The reaction between antigen and antibody, and antibody and enzyme-labeled antibody generally reaches its peak at 37°C for 2 to 3 hours. If the time is too short, the sensitivity will decrease; if the time is too long, the adsorbed antigen or complex (at this temperature) may fall off.
7. Antibody
The determination of antibody titer is the same as that of the antigen, using a double array test.
Determination of enzyme substrate reaction time: generally 15min~30min~45min is used. The standard positive serum can also be used as the standard, and its OD value can be measured at any time. When the specified OD value is reached, the reaction is terminated.
