This article analyzes the dual indirectness of biological function evaluation and detection analysis methods from a holistic perspective, and takes the most basic biological function experiments and ligand receptor binding related experiments as examples to introduce the general process of using ELISA technology competition method for ligand receptor binding related experimental methods. It distinguishes the specific applications and preliminary evaluation of method quality from the two aspects of using samples for affinity comparison and quantitative detection as the purpose, and discusses the general principles of using the same and different detection techniques for functional and quantitative experiments.

The Dual Indirectness of Biological Function Evaluation and Detection Analysis Methods

Biological detection methods are essential tools in the field of life science research and drug development to study the interactions and their effects between bioactive substances, or to quantify the interactions between bioactive substances. Unlike instrument analysis methods such as chromatography-mass spectrometry and Raman spectroscopy, biological detection methods are often indirect, and their intermittency results in lower reliability and stability compared to instrument analysis methods. Understanding the indirectness of biological detection methods from a macro perspective is one of the key factors in establishing a stable biological detection method.

As shown in the figure below, the instrument analysis method (a) studies the characteristics directly related to the sample itself, such as the molecular weight and charge of the sample; On the other hand, the biological detection method (b) first requires the existence or establishment of a test system, followed by the addition of sample reactions to detect changes in the test system caused by the sample. The detection is indirectly related to the sample itself, and the detection target molecular entity is not the sample itself, which is the indirectness of its detection target. For the killing experiment of a certain drug on tumor cells, the test system is for cells, and the change caused is cell death. The target molecular entity for detection is the ATP concentration representing live cells or the LDH concentration representing dead cells.

Most biological detection methods have a complex test system, and changes in the test system caused by the sample also need to be carried out under mild conditions, which makes it difficult to effectively separate the target molecule and detect it using instrument analysis methods. It is necessary to introduce fluorescent labeled antibodies, enzyme-linked antibodies, or enzyme catalyzed substrates to react with the target molecule and convert them into signal values, establishing an indirect connection between the signal values and the target molecule. This is the indirectness of signal transmission in detection. As shown in the figure below, in an ELISA direct method experiment, the sample is an antibody and the detection system is an antigen, causing changes in the target molecule antibody antigen conjugate. The signal value represents the TMB catalytic product, which is indirectly linked to the target molecule antibody antigen conjugate through an enzyme-linked antibody. The quality of an ELISA method is determined by whether the signal is linearly correlated with the target molecule antibody antigen conjugate during the indirect transmission process.

The dual indirectness of biological functional evaluation and detection analysis methods lies in the indirectness of the detection target and the indirectness of the detection signal transmission. Even the simplest biological detection methods, ligand receptor binding assays, have become extremely complex in practical applications due to the indirect nature of these two factors, with many variables and a high risk of false positive or false negative results. A detection method often requires step-by-step experiments in the early stages of method development to optimize its reliability and stability, and to conduct preliminary methodological validation of the established method. Clarifying the target molecules that need to be detected, the correlation pathways between signals and target molecules are the foundation for methodological development, data interpretation, and problem solving.

Development of ELISA Competitive Methodology

Generally speaking, competition law experiments mainly involve three substances: receptors, ligands, and samples that block ligand receptor binding, such as PD1 receptors, PD-L1 ligands, and keyturba antibodies. Antigen, biotinylated antibodies, and sample antibody combinations that block antigen and biotinylated antibodies are also commonly used. In the early stages of competition law development, a test system for receptor ligand interactions was established. The conditions of the test system directly determine the accuracy and measurement range of competitive experiments. Specifically, the development of ELISA competitive methods mainly includes two aspects:

1. Set up multiple negative positive control groups to confirm that the experimental signal values truly represent the target molecule ligand receptor conjugate. The main focus is on whether the enzyme-linked immunosorbent assay (ELISA) plates, enzyme-linked antibodies, and colorimetric reagents used in the indirect detection process interfere with the test system. As shown in the figure below, strictly speaking, when creating an ELISA method, it is necessary to establish a positive reaction group 5 and multiple negative control groups 1-4 to confirm that the signal in the Test system is related to the target molecule. This is a very important and easily overlooked issue. Except for group 5, which can obtain higher positive signals, if other groups obtain stronger positive signals, the data obtained by the entire method is unreliable. Group 3 and Group 5 (yellow) are commonly used control groups in ELISA experiments, but the absence of Group 4 (blue) can make the entire method risky. The experience of developing multiple different ELISA application methodologies by the editor shows that many times the blocking agent cannot achieve the purpose of occupying the blank position of the enzyme-linked immunosorbent assay (ELISA) plate. The reaction ligand or non-specific components in the reaction ligand may directly adsorb onto the ELISA plate, resulting in dose-dependent false positive signals in Group 4. In hybridoma and phage display screening assays, the complexity and diversity of ligand solutions result in the presence of certain negative antibodies or clones that can bind to the enzyme-linked immunosorbent assay (ELISA) plate. The absence of component group 4 significantly increases the frequency of false positive clones. Setting up a comprehensive negative control group can also help identify which reagents in the entire system have undergone cross reactions, identify specific causes, and facilitate reagent replacement.

2. Dose curve of the receptor relative to the reactive ligand. After confirming that the positive signal in the Test system can represent the target detection molecule, a small amount of receptor was coated and the reaction ligand was diluted with a 2-fold gradient at 12-16 points for full curve fitting. The ligand concentration of EC80 or EC50 was selected as the competitive reaction concentration. As shown in the figure below, the 100ng/ml receptor was coated with 100ul, and the receptor was diluted in a 2-fold gradient from 100000ng/ml to 0.2ng/ml. Enzyme linked antibodies with different dilution concentrations were used for detection, and the entire dose curve had obvious upper and lower plateau periods. Different enzyme linked concentrations did not have a significant effect on the linear transmission of the signal, and the enzyme linked antibodies played an equal role in amplifying the signal. The EC50 was 12ng/ml and the EC80 was 40ng/ml. 100ul of 100ng/ml receptor can be optionally coated, with EC80 as the competitive concentration of the ligand at 40ng/ml and an enzyme-linked dilution ratio of 1:600 to obtain a larger signal-to-noise ratio window and increase the measurement range of the method. In the competition law experiment, EC80 represents the ligand occupying 80% of the binding site of the receptor in the Test system. In this relative state, the sample is more sensitive to blocking the response of the ligand and receptor. There are too many receptors, which can provide more binding sites. The binding between the sample and the receptor will not affect the binding between the ligand and the receptor; There are too many ligands, and after adding the sample, there is relatively less competition with the ligands. The vast majority of ligands still bind to the receptor, and in both cases, the addition of the sample cannot cause significant changes in the Test system (a decrease in ligand receptor conjugates), and the signal changes are not significant.

After establishing a complete test system, the dose curve of the competition law is officially established. It mainly includes two aspects of content

1. Mix the ligand with the selected EC80 concentration and the gradient diluted sample (12-16 points diluted with a 2-fold gradient), then add them to the enzyme-linked immunosorbent assay plate coated with good receptors for reaction, and obtain a full curve with obvious upper and lower asymptotes, as shown in the following figure.

2. According to the experimental purpose, select 8 suitable concentration gradient points for the sample for the experiment, simplify the experimental operation process, and preliminarily evaluate the quality of the method. Specifically, in terms of application, it refers to the comparison of affinity levels and quantitative detection. Based on the author's comparative research, it was found that the same curve under the same conditions of ELISA competition method can meet the needs of two aspects with slight adjustment of concentration points. This is a pioneering discovery by the author, and we hope that more peers in the industry can verify it. The following text will provide a preliminary evaluation of two specific application methods.

Preliminary validation of the methodological quality of ELISA competitive assay

When ELISA competitive methods are developed for the purpose of affinity comparison, the IC50 value is often used to evaluate the blocking effect of the tested sample on ligand receptor binding. The smaller the IC50 value, the better the blocking effect of the tested sample. The dose curve developed by affinity competition method should have clear upper and lower plateau periods to obtain relatively stable IC50 values. The methodological validation of biological activity can refer to USP1032, 10331034 for the design of experimental protocols and data analysis. Specifically, for the methodological evaluation of ELISA competitive method, samples with theoretical titers of 156%, 125%, 100%, 80%, and 64% relative to the control were prepared for testing. By comparing the IC50 values of the control and sample, the relative potency of the sample to the measured potency was obtained. The quality of the ELISA competitive method for affinity comparison was preliminarily confirmed by the difference between the measured potency and the theoretical potency. The preliminary methodological validation results of selecting 8 appropriate concentration points based on the full curve of the previous text are shown in the following figure: If multiple people and key reagents are tested in multiple batches in this single experiment, the measured potency obtained will be statistically analyzed to constitute the most important concepts in methodological validation, precision, accuracy, linearity, and range. If the results show that the difference between the measured potency and the theoretical potency in a single experiment is within 5%, it can be preliminarily judged that the ELISA competitive method for affinity comparison has good method quality.

When the ELISA competitive method is developed for quantitative purposes, seven points are selected with theoretical concentration as the X-axis and signal value as the Y-axis for four parameter function fitting to obtain a standard curve. The signal value of the reference substance is then re substituted into the standard curve to obtain the backtesting concentration value. After multiple experimental statistical analyses, the deviation between the backtesting concentration value and the theoretical value determines the detection upper limit, detection lower limit, and measurement range of the standard curve. For more detailed methodological validation, reference can be made to the 2015 edition of the Chinese Pharmacopoeia Part III 9012 Guidelines for Validation of Quantitative Analysis Methods for Biological Samples or the latest version of the Guidance for Industry Bioanalytical Method Validation by the US FDA. As shown in the figure below, within the concentration range of 12.5ng/ml-10000ng/ml, the deviation between the measured concentration and the theoretical concentration of the reference substance is within 20%, indicating that the method has good quality. It should be noted that the standard curve of this quantitative method only removes the lowest concentration point during the upper plateau period from the dose curve comparing affinity sizes. The author's extensive practice has shown that the clear plateau points of the standard curve in quantitative methods can seriously interfere with the quantitative accuracy of the standard curve.

Summary and outlook

This article provides an overview of the general principles of functional and quantitative experiments through the development and preliminary validation of ELISA competitive methodology, including the following steps:

1. Given the dual indirectness of biological methods, identify the target molecules for detection, establish multiple positive and negative control groups, and clarify the linear relationship between signal values and detection target molecules;

2. Establish a test system and conduct ligand receptor binding experiments to find the appropriate relative concentration of ligands and receptors to obtain the sensitivity of the sample to the test system;

3.Add gradient diluted samples into the Test system and plot the full dose curve of the samples relative to the Test system;

4. Guided by the experimental purpose, different concentration points were selected for the purpose of comparing affinity and quantifying large molecules, and the quality of the method was preliminarily evaluated through a compliant methodological evaluation procedure.

The ELISA direct method and ELISA competitive method have the same methodological quality evaluation procedures and standards, namely the methodological quality evaluation procedure and standard USP103210331034 for affinity size comparison; The quality evaluation procedures and standards for quantitative analysis methodology in the 2015 edition of the Chinese Pharmacopoeia, Part III, 9012, Guidelines for Validation of Quantitative Analysis Methods for Biological Samples, or the latest version of the Guidance for Industry Bioanalytical Method Validation by the US FDA. The ELISA competition method can be used for both affinity comparison and large molecule quantification of control concentration points under the same experimental conditions. This is different from my previous articles on the overview of ELISA technology applications and preliminary analysis of methodology development. The ELISA direct method has different experimental conditions for two different applications, but all four applications of the two methods can be verified to meet the standard requirements. It can only be said that practice makes perfect. Regardless of whether it is a white cat or a black cat, the one that can catch mice is a good cat.

In all in vitro functional experiments, the sample causes changes in the detection system and is displayed in the form of signals, which should be the most common principle of in vitro biological experimental methods. Only with binding can there be function. Function is the accurate quantification of changes (response markers) caused by drugs. Understanding the application of binding and quantification in ELISA experiments, as well as the quality evaluation standards for methods, is of great reference significance for the development and evaluation of all functional experiments. There are many possibilities in ELISA.

reference material:

1. Guidelines for Validation of Quantitative Analysis Methods for Biological Samples in Part 9012 of the 2015 Chinese Pharmacopoeia

2. Guidance for Industry Bioanalytical Method Validation。U.S.FDA

3. USP 1032 DESIGN AND DEVELOPMENT OF BIOLOGICAL ASSAYS

4. USP 1033 BIOLOGICAL ASSAY VALIDATION

5. Wu G. Assay Development: Fundamentals and Practices[J]. Cell & Tissue Research, 2010, 348(2):309-313.

6. Difficulties in publishing confirmatory studies in preclinical research: Sun Xuejun Science Network

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