Affinity refers to the dissociation equilibrium constant between antibodies and target antigens, which is a very important biological property. In the quality control process, the ELISA method is often used to evaluate the affinity of different batches or long-term stability samples. Affinity is generally an absolute value, but due to the complexity of biological experiments, the replacement of material batches may affect the accuracy of sample affinity detection. A reference substance with a default quality attribute that does not change is often introduced as a reference substance. The sample to be tested and the reference substance are tested together to offset the deviation caused by the material, and the affinity (relative binding activity) of the sample to be tested relative to the reference substance is calculated. Antibody EC50 refers to the half-maximum effective concentration, which is used to measure the affinity of the antibody. In the functional study of antibodies, the EC50 value indicates the concentration required for the antibody to achieve its maximum effect under specific experimental conditions. A lower EC50 is better, indicating that the antibody has a higher affinity for the target substance.

I. Calculation of EC50 value by saturation concentration method

Using the saturation concentration method (R represents receptor or antigen, L represents ligand or antibody), if the affinity of ligand relative to receptor is to be determined, L is graded diluted in the presence of a small amount of R, and the concentration of R-L complex is detected. When the concentration of R-L complex accounts for half of the total R concentration, the concentration value corresponding to L (EC50) is the KD value of L relative to R. RL complex/R, i.e., B value, can be replaced by the detected signal value. The functional relationship between graded diluted L and signal satisfies the following formula. Only under this specific experimental condition can the EC50 value be equivalent to the KD value.

There are several key points in the determination of KD value by saturation concentration method, i.e., two molecules interacting with each other, R should be in a small amount, L should be graded diluted continuously, and the maximum signal response value (all R binding sites are occupied by L) should be obtained, i.e., the plateau phase. Under this condition, when half of R is occupied, the required L concentration (EC50) is the KD value. The calculation equation is as follows:

Figure 1 Calculation equation

II. Antibody EC50 determination steps

1. Select the appropriate antigen: select the specific antigen that binds to the antibody according to the research purpose.

2. Select the appropriate experimental system: you can choose antigens expressed on the cell surface or synthetic antigens, etc.

3. Prepare antigen solution: prepare a series of antigen solutions according to a certain concentration sequence.

4. Operating platform setting: use professional biotechnology equipment and software to set up the experiment.

5. Antibody and antigen binding: react the antibody with the antigen solution and let it reach equilibrium after a certain period of time.

6. Reaction termination and separation: terminate the reaction by appropriate methods and separate the unbound antibody from the antigen.

7. Signal detection: use appropriate detection methods, such as enzyme-linked immunosorbent assay (ELISA), to determine the number of bound antibodies.

8. Data analysis: draw a curve based on the experimental results and calculate the EC50.

III. Notes on antibody affinity testing

1. The standard dose curve fitting generally uses four-parameter fitting, which requires obvious upper and lower plateaus (A, D values), and the slope is generally between 0.8-1.2 (B value). EC50 (C value) is the affinity value of the antibody relative to the antigen.

2. The enzyme-linked antibody effect is excessive, and the dilution of the enzyme-linked antibody needs to be compared and selected at different dilutions. For the enzyme-linked antibody of the monoclonal antibody component, if the enzyme-linked antibody of the high and low dilutions can produce the same dose curve, it means that the enzyme-linked antibody is excessive and the signal transmission of ELISA has not produced deviation. For the enzyme-linked antibody of the polyclonal antibody component, the judgment of its dilution is more complicated. It can be roughly calculated that the molar concentration of the enzyme-linked antibody is more than 5 times higher than the molar concentration of the antigen on the coating. When the concentration of the enzyme-linked antibody is small, the enzyme-linked antibody is captured under the conditions of high doses and different concentrations of antibodies, and the curve has a false upper platform.

3. If you need to reduce the non-specific adsorption of high-concentration antibodies (100000ng/ml) to the enzyme-labeled plate during the reaction, it is recommended to choose a hydrophobic enzyme-labeled plate (polysorp) during the coating process. The concentration of the coated antigen needs to be a small amount of 0.2-1ug/ml. The specific situation needs to be judged and optimized according to the affinity of the antigen and antibody. Generally speaking, the stronger the affinity, the lower the required antigen coating concentration.

4. For absorbance value substrates and fluorescent substrates, the signal value detected by the instrument is proportional to the color development time, and it is advisable to control it within 10-30 minutes. It is necessary to confirm the signal linear range of the detection instrument in advance. For example, the signal linear range of the absorbance value of a general microplate reader is 0-3, and the absorbance value is nonlinear between 3-4. When developing color, the signal value should be controlled not to exceed 3. When the signal value exceeds the linear range, the curve has a false upper platform.

Tek Biotech is committed to affinity detection research and can provide customers with professional and effective pre-sales technical consultation and accurate data support, providing strong guarantees for customers' experimental projects. At present, there are many commonly used methods for detecting affinity, but most of them either rely on expensive equipment (surface plasmon resonance), are not generally applicable (fluorescence modification, dialysis), or require modified antigens (radioactive precipitation labeled antigens). The competitive ELISA method for determining affinity is an exception, which only relies on a small amount of purified antigen and antibody for affinity detection.