I talked to everyone in the last episode of Xiao Ao ClassroomELISA Methodology Development Practice Edition - Quantitative Method Development for Biomacromolecule Detection.
In this issue, Xiao Ao will share another important topic with you: "ELISA Methodology Development Practice Edition - Affinity Determination Method Development".

1、 Preface

At present, the methods used to determine the affinity between receptor ligands, that is, the dissociation equilibrium constant, mainly include:

1. Thermodynamic determination method
2. Dynamics determination method
3. Saturation concentration method

The kinetic determination method and saturation concentration method are more commonly usedDynamics determination method(such as Biacore)Involving relatively expensive instrument investment,The saturation concentration determination method is commonly used by the vast majority of biopharmaceutical companies in China.

ELISA is the mainstream mode of saturation concentration assay for affinity determination, commonly used for activity evaluation of antigen proteins or antibody protein levels and stability evaluation of antibodies.
ELISA for affinity determination often does not have a commercially available kit and requires methodological development by laboratory personnel.

Due to the lack of education in biotechnology and insufficient accumulation of new drug development companies in its application, an ELISA affinity assay method that can be validated through methodology is a difficult task for many practitioners, and there is a high possibility of errors such as new account transmission errors or biases.
One of the misconceptions is the development of an ELISA affinity assay method using quantitative ELISA experimental conditions.

This article starts with the basic principles of affinity determination methodsDescribe the basic principles of affinity determination using saturation concentration method, outline the dose relationship between antigen and antibody in the development process of ELISA method, as well as several errors that may occur when ELISA is used for affinity determination method, and introduce the general affinity determination scheme and preliminary methodology development scheme for everyone.

2、 Basic concepts of affinity

We will explain what affinity is by analyzing several chemical reaction cases.

例子A
Hydrogen and oxygen generate water under ignition conditions.
When the reaction stops, if the molar concentration of hydrogen in the reaction environment happens to be twice that of oxygen, both are consumed and there is only water in the reaction environment; If the molar concentration of hydrogen is greater than twice that of oxygen, oxygen is consumed and only hydrogen and water are present in the reaction environment; When the molar concentration of hydrogen is less than twice that of oxygen, hydrogen is consumed and there is only oxygen and water in the reaction environment.
This is the basic knowledge about chemical reactions. The result of a chemical reaction is that the reactants can turn into products, that is, the reaction can continue until the end. A reaction with the process and outcome characteristics of such a reaction is called an irreversible reaction.

例子B
PD1 antigen（CD279）And PD1 antibody(such as PE)mbrolizumab）React at room temperature and pressure.
At the end of the reaction, if the molar concentration of CD279 in the reaction environment is 200nM and the molar concentration of pembrolizumab is 100nM, under ideal conditions, the concentration of pembrolizumab-CD279 complex is 97.86nM, the molar concentration of CD279 is 4.28nM, and the molar concentration of pembrolizumab is 2.14nM;
If the molar concentration of CD279 in the reaction environment is 100nM and the molar concentration of pembrolizumab is 200nM, under ideal conditions, the concentration of pembrolizumab-CD279 complex is 49.81nM, the molar concentration of CD279 is 0.38nM, and the molar concentration of pembrolizumab is 159.19nM.

Example C
PD1 antigen（CD279）And PDL1 antigen（CD274）React at room temperature and pressure.
If the molar concentration of CD279 in the reaction environment is 200nM and the molar concentration of CD274 is 100nM, under ideal conditions at the completion of the reaction, the concentration of CD274-CD279 complex is 2.35nM, the molar concentration of CD279 is 197.65nM, and the molar concentration of CD274 is 97.65nM;
If the molar concentration of CD279 in the reaction environment is 100nM and the molar concentration of CD274 is 200nM, under ideal conditions at the completion of the reaction, the concentration of CD274-CD279 complex is 2.35nM, the molar concentration of CD279 is 97.65nM, and the molar concentration of CD274 is 197.65nM. The vast majority of CD279 and CD274 did not participate in the reaction.
In cases B and C, both antigens and antibodies(Ligands and receptors)The combination reaction involves both the positive reaction process of generating complexes and the reverse reaction process of complex dissociation reaction to generate antigens and antibodies. The ultimate result is the coexistence of antigens, antibodies, and antigen antibody complexes.

This reaction is calledreversible reaction.
When the rates of forward and reverse reactions in a reversible reaction are equal, the concentration of antigen, antibody, and antigen antibody complex in the reaction system no longer increases or decreases, and the reversible reaction is completed. The molar concentrations of each component in the reaction system are in dynamic equilibrium.

affinity(Affinity)It is a characteristic parameter of the relative state between antigens, antibodies, and antigen antibody complexes in reversible reaction processes. Its more specialized and technical name is the dissociation equilibrium constant KD, expressed in mol/L(Consistent with concentration units).

The smaller the affinity value, the stronger the affinity, and the strength of the affinity determines the relative amount of reversible reaction components at the end of the reaction.

Examples B and C are also similar to 200nM CD279（PD1）Reaction, antibody pembrolizumab(PD1 antibody)Relative affinity to CD279 is 0.4nM, ligand CD274（PDL1）Compared to the affinity of 8200 nM for CD279, the pembrolizumab-CD279 complex generated by the final reaction is 97.86 nM, while the CD274-CD279 complex is only 2.35 nM, a difference of 42 times between the two.

Unlike irreversible reactions, in addition to the molar concentration of antigens and antibodies, antigens and antibodies(Ligands and receptors)The affinity between the two will have an impact on the amount of products produced in reversible reactions.

3、 Do you know the basic principles of ELISA methodology development?

For an overview of affinity determination methods and principles, readers can refer to the Affinity Evaluation section of Biological Functional Screening and Evaluation Techniques.
This article mainly summarizes the saturation concentration method.
Rrepresentreceptororantigen，Lrepresentligandorantibody.
Using the saturation concentration method, if the affinity of the ligand relative to the receptor is to be determined, in the presence of a small amount of R, L is diluted in a gradient to detect the concentration of the R-L complex. When the concentration of the R-L complex accounts for half of the total R concentration, the corresponding concentration value of L（EC50）The KD value of L relative to R.
The RL complex/R or B value can be replaced by the detected signal value. The functional relationship between the gradient diluted L and the signal satisfies the following formula, and only under specific experimental conditions can the EC50 value be equivalent to the KD value.
There are several key points in determining KD values using the saturation concentration method,The two molecules that interact with each other, R needs a small amount and L needs to undergo continuous gradient dilution to obtain the required signal response value(All binding sites of R are occupied by L)During the platform period, under these conditions, the required L concentration is required to occupy half of R（EC50）For KD value.
The saturation concentration method mainly measures the signal value representing the RL complex, and the measurement methods can be flow cytometry, isotope labeling, fluorescence polarization, or ELISA.

4、 Common methods for developing ELISA methodology for affinity determination

layout

General operating procedures

01Preparation of enzyme label plate: take antigen with a concentration of 100 ug/ml, dissolve it at room temperature, mix it well and dilute it with coating solution to 0.2 ug/ml, 0.5 ug/ml and 1 ug/ml according to the following steps, add 100 ul/well according to the sample adding layout table, and add them into enzyme label Flat noodles respectively(Note 1)Add coating solution as a control for coating antibodies and overnight at 2-8 ℃;
02Wash the board three times with detergent, pat dry, and add 300ul/well sealing solution(Note 2)Seal at room temperature for 1 hour; Wash the board with detergent three times, pat dry and set aside for later use;
03Dilute the antibody in a 3-fold gradient starting from 100000ng/ml according to the layout（Note 3）According to the layout, add 100ul/hole into the microporous plate;
04put inOhhaus microplate oscillator (ISLDMPHDG)Internally, set the temperature to 37 ℃ and oscillate at 600rpm for 1 hour;

05Wash the board three times with detergent and pat dry;
06Dilute enzyme-linked antibodies to the appropriate concentration（Note 4）, inOhhaus vortex analyzer（VXMNFS）upMix well, 100ul/hole;

07Put it into a microplate oscillator and set it to oscillate at 37 ℃ and 600rpm for 1 hour;
08Wash the board 4 times with detergent and pat dry;
09Take the colorimetric solution corresponding to the enzyme-linked antibody, take it out and equilibrate it to room temperature 20 minutes before use, and mix it evenly on a vortex mixer;
10Add color developer at a rate of 100 ul/hole using 8 gun rows;
11Place the color developing solution at room temperature in the dark for 10-30 minutes（Note 5）；
12Use 8 guns to add termination solution at 100 ul/well to terminate the reaction(Depending on the substrate, this step may not be necessary)；
13Measure signal values using an enzyme-linked immunosorbent assay (ELISA) reader（Note 6）；
14Result Analysis（Note 7）.

Thoughtful little note

Note1：If it is necessary to reduce the high concentration of antibodies during the reaction process（100000ng/ml）For non-specific adsorption of enzyme-linked immunosorbent assay (ELISA) plates, it is recommended to choose hydrophobic ELISA plates during the coating process（polysorp）The antigen concentration of the coating needs to be a small amount of 0.2-1ug/ml, and the specific situation needs to be optimized based on the affinity of the antigen antibody. Generally speaking, the stronger the affinity, the lower the required antigen concentration.
Note2：Sealing liquid may not necessarily have a sealing effect, and experimental verification is needed(Group without antigen encapsulation)Generally speaking, the smaller the molecular weight of the sealing agent, the better the effect.
Note3：In the early stages of methodological development, the concentration range of the standard curve can be widened to 100000ng/ml-0.1ng/ml to obtain a full curve with clear upper and lower plateau periods. When the affinity between antigen and antibody is strong, the gradient concentration can be reduced, and the dilution factor can be reduced to 2 times during methodological optimization.
Note4：The effect of enzyme-linked antibodies is excessive, and the dilution of enzyme-linked antibodies needs to be compared and selected at different dilutions. For enzyme-linked antibodies with monoclonal antibody components, if the enzyme-linked antibodies with high and low dilutions can produce the same dose curve, it indicates that there is an excess of enzyme-linked antibodies and there is no deviation in the signal transmission of ELISA.
For enzyme-linked antibodies with multiple antibody components, the determination of their dilution is relatively complex. It can be roughly calculated that the molar concentration of the enzyme-linked antibody should be more than 5 times higher than the molar concentration of the antigen on the coating. When the concentration of enzyme-linked antibodies is low, the enzyme-linked antibodies are captured under high doses and different concentrations of antibody conditions, and the curve has a false plateau.
Note5：For substrates with absorbance values 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 linear range of the signal of the detection instrument in advance. For example, the linear range of the absorbance value of a general enzyme-linked immunosorbent assay (ELISA) is 0-3, and the absorbance value between 3-4 is non-linear. During color development, the signal value should be controlled not to exceed 3. When the signal value exceeds the linear range, the curve will have a false plateau.
Note6：Different substrates require different instruments for detection, including absorbance readings, fluorescence readings, and chemiluminescence readings. During the substrate selection process, pay attention to whether the laboratory's enzyme-linked immunosorbent assay (ELISA) reader has this type of reading function.
Note7：The standard dose curve fitting generally uses four parameter fitting, which requires a clear upper and lower plateau period(A, D values)The slope is generally between 0.8-1.2(B value)between, EC50（C value)It is the affinity value of the antibody relative to the antigen.

V. Summary

According to the saturation concentration method, when using ELISA for affinity determination, a clear four parameter S-shaped curve is required, which requires a signal curve that gradually changes with antibody concentration.
There is a clear upper and lower plateau period at low and high concentrations of antibodies, with half of the signal value in the upper plateau period corresponding to the antibody concentration（EC50）The value refers to the KD value of antigen antibody affinity.
Due to the limited linear range of the detection signal of the instrument, when the signal exceeds the linear range, it will cause the curve to enter a false plateau period, resulting in inaccurate measured affinity.
The antigen concentration of the coating directly determines the signal value of the signal,Therefore, when using ELISA for affinity determination, the concentration of the coated antigen should be minimized as much as possible to obtain appropriate signal values; When the concentration of enzyme-linked antibodies is too low, it may also cause the curve to enter a false plateau period.

In the development process of ELISA methodology, the linear transmission of signals due to excess enzyme-linked antibodies requires experimental verification.
When the signal cannot obtain an effective plateau period, it indicates that the affinity between antigens and antibodies is too weak or there is too much antigen coating, requiring a higher concentration of antibodies to capture all antigens and reach the plateau period.
Due to the limitation of ELISA sensitivity, some affinity is weak（>100nM）In the antibody antigens, the ELISA method cannot obtain an effective plateau period or measure the affinity value.
When evaluating antigen activity or antibody activity using ELISA affinity assay, the EC50 obtained is not necessarily the smaller the better, and the signal enters a false plateau period(Reached the detection limit of the instrument, with low enzyme-linked concentration)All of them will cause the EC50 to decrease, resulting in incorrect affinity data, which is also a common fallacy in ELISA affinity determination.
Nowadays, many suppliers of antigen proteins have such misconceptions in their evaluations of antigen activity.

The affinity between antigens and antibodies is determined by their structures and interactions,It may be 0.1 nM, 1 nM, or 1000 nM. To evaluate the quality of the ELISA affinity assay method, it is necessary to check whether the EC50 value matches the affinity value. The affinity value can be verified through various methods such as thermodynamic assay, kinetic assay, and saturation concentration assay using cell flow cytometry.
On the other hand, when an ELISA method does not have signals such as entering a false plateau period(Reached the detection limit of the instrument, with low enzyme-linked concentration)When there is a fallacy, its EC50 value can truly reflect the numerical value of affinity.

The use of ELISA for methodological development requires clear application purposes, macromolecular quantification, and affinity determination.
There will be significant differences in the relative dose concentration of antigens and antibodies for different application purposes, with different dose curves,Readers can compare and study the differences by referring to the previous article 'ELISA Methodology Development Practice Edition - Quantitative Method Development for Biomacromolecule Detection'.

That's all for this sharing, see you next time!

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