1. Selection of solid phase carriers There are many types of carriers, including cellulose, cross-linked dextran, staphylococcus polystyrene, polyacrylamide, and the like. There may be a pit plate, a test tube, a bead or the like from the form of use.
Polystyrene pits are the most widely used carrier. The polystyrene plastic microtiter plate has good performance in adsorbing protein, simple operation and small dosage, and is suitable for mass inspection.
Due to the insufficiency of the process of polystyrene, the batches vary greatly. Therefore, screening must be performed before the ELISA is performed. Inspection Method:
(1) Adsorption performance test: first add antibody coating, then add the same dilution of the enzyme-labeled antibody, and finally add substrate, color, OD value, find the total average OD value, and then find each adjacent two holes The average value of OD is within ±10% of the total mean value. If the difference between the OD value of the middle hole and the surrounding hole is too large, or the difference between the OD values ​​of one side and the other side is large, it is unqualified.
(2) Contrast positive serum and negative serum to observe whether there is a significant difference. If the OD values ​​of the two are different by more than 10 times, they are qualified.
Board processing. The new board is generally not processed and can be applied by rinsing with distilled water. The board is used once and is wasted. However, many experimental workers believe that the ultrasonic treatment, cleaning solution Tritonx100, 20% ethylene glycol treatment can still be applied. However, when the color of the blank control is darker and the color of the positive sample is not ideal, it should be discarded.
2. Adsorption conditions of the carrier The adsorption of the carrier is physical adsorption. The amount of adsorption depends on pH, temperature, protein concentration, ionic strength, and adsorption time.
The preferred adsorption conditions are: ionic strength of 0.05Mol / L ~ 0.10Mol / L, pH 9.0 ~ pH 9.6 carbonate buffer, protein concentration of 1μg / ml ~ 100μg / ml, 4 ° C overnight or 37 ° C 3h.
3. The concentration of the enzyme-labeled antibody is determined by adding a sufficient amount of antibody to the well of the polystyrene plate, incubating for a certain period of time, washing, diluting the enzyme-conjugated compound, adding 2 wells per dilution, incubating and rinsing Add substrate color and color. The OD value is plotted on the ordinate, and the dilution of the enzyme-labeled conjugate is plotted on the abscissa to prepare a curve. When the OD value is 1, the corresponding dilution of the enzyme-labeled antibody is the optimal dilution of the enzyme-labeled antibody.
This dilution refers to the optimum dilution under these conditions, and the other conditions are not optimal. For example, a 1:400 dilution of the enzyme-labeled antibody for 6 h can be the same as the incubation of 1..6 400 dilution for 24 h. Therefore, once the conditions are determined, do not change to ensure the repeatability and relative accuracy of the results.
The dilution of the optimal enzyme-labeled antibody can be used as a working concentration, and can also be increased by half to one titer, but cannot be increased too high, otherwise non-specific color development is increased.
The titer of the enzyme-labeled antibody reflects the quality of the enzyme-labeled antibody, and it is also possible to compare the advantages and disadvantages of the enzyme-labeled combination. Some materials reported that 1..320 titers are qualified, and more than 1..1 000. 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:
(1) Antigen requirements:
Antigens used in ELISA must use relatively pure antigens, and if they contain other impurities, will compete with the antigen for a limited position on the solid support. Antigens used in other serological reactions may not be suitable for ELISA experiments and must be tested so that the antigen must be firmly adsorbed onto the carrier without loss of its immunological activity and results in regular repeats. In addition, after adsorbing the carrier, minimal non-specific adsorption is applied to the various reagents added, that is, the difference between the binding to the negative and positive serum is large.
(2) Determination of antigenic titer A single square or two-layer array test can be used. 1 Single square matrix test: coated with different dilutions of antigen on the ELISA plate, added with normal 1:200 dilution of positive serum, then added enzyme-labeled antibody, color development, OD value, when the OD value is 1.0 The corresponding antigen concentration is used as the potency; 2 the two-layer array test is more accurate, it can measure the optimal concentration of the antigen and can measure the optimal concentration of the antibody. The antigen-antibody is diluted to different concentrations to carry out the enzyme-labeled antibody reaction, and the corresponding antigen dilution which is the difference in the light absorption value of the negative and positive serum having the highest serum dilution ratio is the use titer of the antigen.
The solid phase carrier of the adsorbed antigen is stably stored by lyophilization or drying, and remains inactive for several months.
5. The cleaning solution is generally used in 0.01 Mol / L pH 7.2 PBS Tween buffer. Tween is a polyoxyethylene sorbitan fatty acid ester, which is a nonionic surface tension substance and is often used as a cosolvent. The Tween number depends on the type of fatty acid to which the sorbitol is bound. Tween 20 is combined with lauric acid, Tween 40 is combined with palmitic acid, Tween 60 is combined with stearic acid, and Tween 80 is combined with oleic acid. Tween 20 is usually added to the buffer as a wetting agent to reduce non-specific adsorption. 1% bovine serum albumin (or 10% calf serum or ovalbumin) can also be added to PBS buffer, especially after antigen coating, and then re-coated with bovine serum albumin buffer to occupy the pores. The remaining position, in order to reduce non-specific reactions.
6. Reaction time The reaction between the antigen and the antibody, the antibody and the enzyme-labeled antibody generally reaches a peak at 37 ° C for 2 h to 3 h. The time is too short, the sensitivity is reduced, the time is too long, and the adsorbed antigen or complex (at this temperature) may fall off.
7. Antibody The titer of the antibody was determined in the same manner as the antigen.
Determination of the reaction time of the enzyme substrate: generally 15min ~ 30min ~ 45min. The standard positive serum can also be used to determine the OD value at any time, and when the specified OD value is reached, the reaction is terminated.
Polystyrene pits are the most widely used carrier. The polystyrene plastic microtiter plate has good performance in adsorbing protein, simple operation and small dosage, and is suitable for mass inspection.
Due to the insufficiency of the process of polystyrene, the batches vary greatly. Therefore, screening must be performed before the ELISA is performed. Inspection Method:
(1) Adsorption performance test: first add antibody coating, then add the same dilution of the enzyme-labeled antibody, and finally add substrate, color, OD value, find the total average OD value, and then find each adjacent two holes The average value of OD is within ±10% of the total mean value. If the difference between the OD value of the middle hole and the surrounding hole is too large, or the difference between the OD values ​​of one side and the other side is large, it is unqualified.
(2) Contrast positive serum and negative serum to observe whether there is a significant difference. If the OD values ​​of the two are different by more than 10 times, they are qualified.
Board processing. The new board is generally not processed and can be applied by rinsing with distilled water. The board is used once and is wasted. However, many experimental workers believe that the ultrasonic treatment, cleaning solution Tritonx100, 20% ethylene glycol treatment can still be applied. However, when the color of the blank control is darker and the color of the positive sample is not ideal, it should be discarded.
2. Adsorption conditions of the carrier The adsorption of the carrier is physical adsorption. The amount of adsorption depends on pH, temperature, protein concentration, ionic strength, and adsorption time.
The preferred adsorption conditions are: ionic strength of 0.05Mol / L ~ 0.10Mol / L, pH 9.0 ~ pH 9.6 carbonate buffer, protein concentration of 1μg / ml ~ 100μg / ml, 4 ° C overnight or 37 ° C 3h.
3. The concentration of the enzyme-labeled antibody is determined by adding a sufficient amount of antibody to the well of the polystyrene plate, incubating for a certain period of time, washing, diluting the enzyme-conjugated compound, adding 2 wells per dilution, incubating and rinsing Add substrate color and color. The OD value is plotted on the ordinate, and the dilution of the enzyme-labeled conjugate is plotted on the abscissa to prepare a curve. When the OD value is 1, the corresponding dilution of the enzyme-labeled antibody is the optimal dilution of the enzyme-labeled antibody.
This dilution refers to the optimum dilution under these conditions, and the other conditions are not optimal. For example, a 1:400 dilution of the enzyme-labeled antibody for 6 h can be the same as the incubation of 1..6 400 dilution for 24 h. Therefore, once the conditions are determined, do not change to ensure the repeatability and relative accuracy of the results.
The dilution of the optimal enzyme-labeled antibody can be used as a working concentration, and can also be increased by half to one titer, but cannot be increased too high, otherwise non-specific color development is increased.
The titer of the enzyme-labeled antibody reflects the quality of the enzyme-labeled antibody, and it is also possible to compare the advantages and disadvantages of the enzyme-labeled combination. Some materials reported that 1..320 titers are qualified, and more than 1..1 000. 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:
(1) Antigen requirements:
Antigens used in ELISA must use relatively pure antigens, and if they contain other impurities, will compete with the antigen for a limited position on the solid support. Antigens used in other serological reactions may not be suitable for ELISA experiments and must be tested so that the antigen must be firmly adsorbed onto the carrier without loss of its immunological activity and results in regular repeats. In addition, after adsorbing the carrier, minimal non-specific adsorption is applied to the various reagents added, that is, the difference between the binding to the negative and positive serum is large.
(2) Determination of antigenic titer A single square or two-layer array test can be used. 1 Single square matrix test: coated with different dilutions of antigen on the ELISA plate, added with normal 1:200 dilution of positive serum, then added enzyme-labeled antibody, color development, OD value, when the OD value is 1.0 The corresponding antigen concentration is used as the potency; 2 the two-layer array test is more accurate, it can measure the optimal concentration of the antigen and can measure the optimal concentration of the antibody. The antigen-antibody is diluted to different concentrations to carry out the enzyme-labeled antibody reaction, and the corresponding antigen dilution which is the difference in the light absorption value of the negative and positive serum having the highest serum dilution ratio is the use titer of the antigen.
The solid phase carrier of the adsorbed antigen is stably stored by lyophilization or drying, and remains inactive for several months.
5. The cleaning solution is generally used in 0.01 Mol / L pH 7.2 PBS Tween buffer. Tween is a polyoxyethylene sorbitan fatty acid ester, which is a nonionic surface tension substance and is often used as a cosolvent. The Tween number depends on the type of fatty acid to which the sorbitol is bound. Tween 20 is combined with lauric acid, Tween 40 is combined with palmitic acid, Tween 60 is combined with stearic acid, and Tween 80 is combined with oleic acid. Tween 20 is usually added to the buffer as a wetting agent to reduce non-specific adsorption. 1% bovine serum albumin (or 10% calf serum or ovalbumin) can also be added to PBS buffer, especially after antigen coating, and then re-coated with bovine serum albumin buffer to occupy the pores. The remaining position, in order to reduce non-specific reactions.
6. Reaction time The reaction between the antigen and the antibody, the antibody and the enzyme-labeled antibody generally reaches a peak at 37 ° C for 2 h to 3 h. The time is too short, the sensitivity is reduced, the time is too long, and the adsorbed antigen or complex (at this temperature) may fall off.
7. Antibody The titer of the antibody was determined in the same manner as the antigen.
Determination of the reaction time of the enzyme substrate: generally 15min ~ 30min ~ 45min. The standard positive serum can also be used to determine the OD value at any time, and when the specified OD value is reached, the reaction is terminated.
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