The ELISA procedure can be roughly divided into three processes. These are ELISA protocol design, ELISA initial data interpretation, and ELISA results calculation.
A well-thought-out and perfect ELISA experimental protocol design require the rational arrangement of various experimental factors and strict control of experimental errors, thus avoiding the waste of human, material, and financial resources caused by blindly conducting experiments.
Usually, we recommend that the first two columns of the 96 microtiter plate used for ELISA experiments be set up as standard spotting wells, while setting up 8 gradient concentrations, 2 columns of compound wells, and adding the standard working solution from low to high concentrations, sequentially from the bottom to the top of the enzyme standard plate.
The last 10 columns of the microtiter plate can be set up as the sample spotting wells for the samples to be tested, and generally set up 2 duplicate wells. According to the experimental requirements, a normal group, model group, treatment group, etc. can be set.
Precision: The precision of an Enzyme-linked immunosorbent assay reflects the reproducibility of the measured data. Precision is generally expressed by the coefficient of variation CV (CV = standard deviation / mean × 100%). In general, the CV of both the standard and the sample OD should be ≤ 10%.
OD value: The OD value of the blank well of ELISA is ≤ 0.1 (sandwich method). The maximum OD of the standard is ≥1.2. The OD of the sample should fall within the range of the standard curve.
Linear range: R2 ≥ 0.99 for linear regression correlation coefficient of the standard curve.
As the last step of the ELISA experiment, the calculation of the assay results is very important for the whole experiment.
We take the sandwich method as an example to introduce the ELISA experiment result calculation.
ELISA samples usually have to set up 1~2 duplicate wells for testing, and the average value of absorbance of standards and experimental group samples are derived respectively. The detection value of a single sample should not exceed 20% of the average value.
The average value of OD (absorbance) of ELISA samples should be subtracted from the average value of OD when the concentration of standard is 0.
Establish the standard curve: fit a logistic function curve of four parameters on a logarithmic coordinate plot (standard concentration on the X-axis and the corresponding OD value on the Y-axis). Recommend using origin software.
Step 1: Enter the standard concentration and OD value
Step 2: Select the type of curve to be fitted - non-linear curve fitting
Step 3: Select Statistics and Logistic, check "Find X from Y"
Step 4: View ELISA standard curve information (e.g. parameter values, R-Square, etc.)
Step 5: Modify the title of the horizontal and vertical coordinates and set the axis type to logarithmic
Double-click the ELISA fit graph in step 4 and edit it. Double-click the title of the modified horizontal and vertical coordinates, double-click the X-axis and Y-axis, set the coordinate type to "Log10", and set the horizontal and vertical coordinate range.
Step 6: Calculate the sample concentration from the standard curve
Select the fourth worksheet (Fit NL Find X from Y1), enter the OD value of the sample in the left column, and the corresponding concentration will be automatically generated in the right column.
At least the following points should be mastered when performing ELISA experiments.
The basic composition of the enzyme-linked immunosorbent assay positive control should be the same as that of the assay sample as far as possible. Generally, positive controls are mostly based on a buffer containing a protein protector and adding a certain amount of the substance to be tested. For example, for the determination of human serum as the specimen, the positive control is mostly made of determining patient serum or compound calcium human plasma with a certain amount of standard.
The basic composition of the ELISA negative control should also be consistent with the composition of the test sample as far as possible, and it is best to test first to determine that it does not contain the substance to be tested. For example, when testing human serum specimens, the negative control should be normal human serum; when testing the potency of antibodies in the serum of immunized animals, the negative control should be the pre-immune serum of the animal.
Selection of encapsulated antigens
Encapsulated antigens can be divided into three categories: natural proteins, recombinant proteins, and small molecule antigens. Natural proteins need to be purified to be coated by the direct adsorption method, for antigens containing more impurities can be coated by indirect capture method (first use substances that can react with the target antigen such as antibodies directly adsorbed on the enzyme standard plate, and then make the antigen solid phase by specific reaction). Purified recombinant proteins can generally be directly coated. Small molecule antigens such as peptides and some small molecule organic compounds are often difficult to adsorb directly on the enzyme plate because of their small molecular weight and are generally coupled with unrelated proteins such as BSA, and the couples are adsorbed on the solid phase carrier.
Choice of coating solution
The ELISA coating solution is usually a carbonate buffer pH 9.6, or a phosphate buffer pH 7.2 if the antigen is unstable under alkaline conditions.
Selection of ELISA coating temperature
The ELISA coating temperature is usually 4-8 degrees Celsius overnight or 37 degrees Celsius for 2 hours, we strongly recommend 4-8 degrees Celsius for the maintenance of protein activity.
Selection of ELISA coating concentration
The optimum concentration of ELISA coating varies with the nature of the solid phase carrier and coating, generally, the coating concentration of protein is 1-5ug/ml, to specify the optimum coating concentration for a specific coating antigen needs to be determined by experiment.
Whether closure is necessary depends on the mode of ELISA and the specific experimental conditions. Generally speaking, for double antibody sandwich assay, as long as the enzyme marker is highly active and the operation is thoroughly washed, satisfactory results can be obtained without closure. In contrast, in indirect assays, containment is generally essential.
The commonly used sealants are 0.05%-0.5% BSA, 10% calf serum, 1% gelatin, and 5% skim milk powder, AbMART recommends using 5% skim milk powder, which is inexpensive and has the strong sealing ability, however, 5% skim milk powder is only suitable for short-term use and should not be stored for a long time, so it is less used in the kit.
Dilutions of enzyme conjugates
High concentrations of extraneous proteins (e.g. 1% BSA or 5% skim milk powder) are often added to the diluent to inhibit the non-specific adsorption of enzyme conjugates on the solid phase carrier by competition. A nonionic surfactant capable of inhibiting protein adsorption on the plastic surface, such as Tween 20 (0.05% concentration is preferred), is also generally added.
Proper dilution of enzyme conjugates
The appropriate working concentration of the enzyme conjugate needs to be determined by pre-experiments. Too high a concentration tends to lead to high background, while too low a concentration will lead to a weakening of the positive signal.
The enzyme conjugate should preferably be diluted before use, and the diluted enzyme conjugate should not be stored for a long time, as low concentrations of the enzyme conjugate are highly susceptible to inactivation.
|Negative controls yielded positive results
|1. Contamination of reagents or consumables
2. Problems with plate washing
3. If the problem occurs in the double antibody sandwich method, it is possible that there is a cross-reaction between the coating antibody and the secondary antibody.
4. Use of too many antibodies
|1. Changing reagents, using disposable supplies
2. Change the washing solution with stronger formula, increase the number of plate washing and extend the time of plate washing.
3. Replacement of coated antibody or secondary antibody
4. Reduce the amount of antibodies used.
|The whole board appears high background
|1. Secondary antibody produces non-specific adsorption
2. Chromogenic solution is not fresh
3. The color development reaction is not terminated for too long
4. Contamination of reagents or consumables
5. Non-specific adsorption due to high reaction temperature
Non-specific adsorption due to poor containment conditions
|1. Reduce the amount of secondary antibody used, shorten the reaction time of secondary antibody
2. Use ready-made color development solution
3. Control the reaction time of color development, and terminate the reaction in time
4. Change reagents and use disposable consumables
5. Strictly control the reaction at the optimum temperature
6. Replace the blocking solution with a more powerful blocking solution and extend the blocking time
|Low reaction signal
|1. Inappropriate coating conditions
2 .Inadequate reaction of antigen and antibody
3. Improper formulation of color developing solution
4. Insufficient secondary antibody binding
|1. Increase the concentration of the plate, extend the time of the plate
2. Extend the reaction time to ensure that the reaction is carried out at the optimal temperature and increase the amount of chromogenic substrate
3. Increase the concentration of secondary antibody, extend the reaction time, and replace the secondary antibody with a more effective one.
|Gradient dilution to do ELISA produces jumping hole phenomenon
|1. Uneven heat transfer due to stacking of enzyme plates, differences in reaction temperature of each well
2. Pipette dilution does not maintain continuity
3. Evaporation of reaction solution
4. Uneven plate washing
5. There are debris or water droplets on the bottom of the enzyme plate
|1. Avoid stacking the plates together as much as possible
2. Calibrate the pipette regularly to ensure proper use of the pipette
3. Seal or cover the plates with a seal
4. Make sure the plate washer is working properly and clean the plates when reading