The classic technology of ELISA has many years of development history. There are many types of methods. Competitive ELISA technology is one of the basic types. Like sandwich ELISA technology, it is also applied to drug PK analysis and research; how to be based on competitiveness The principle of ELISA to construct a competitive Assay and grasp the relevant key points in the practice of method establishment is particularly important to the ultimate success of the entire method. The author combined his own practical experience and cases to give a brief explanation in this article for your reference.
Since the ELISA technology was proposed in the 1970s, after decades of development, it has become an indispensable classic technology in the field of biomedicine research and development. The ELISA method has a variety of different types of classification forms. Based on the reaction principle and mode, it can be mainly divided into direct method, indirect method, sandwich method and competition method. Others are based on different molecular marker forms, carrier forms, capture forms or signal generation forms, etc. Methods such as fluorescence method, chemiluminescence method, electrochemiluminescence method, chip-like method and microsphere method are all combinations or derivatives of these four basic methods; among them, the sandwich method is used in the PK of macromolecular drugs such as antibodies and protein drugs. It has been widely used in the field of analysis. For small molecule compounds or peptide drugs with small molecular weights, in addition to LC-MS/MS for their PK analysis, the competitive ELISA method can also exert its unique application value and advantages. Not only that, even proteins and antibodies with larger molecular weights can still be considered for PK analysis using competitive ELISA in practice. Therefore, from the perspective of the range of drug molecule types that can be applied, the competition method is the most promising ELISA method in the field of drug PK analysis.
To date, small molecule drugs account for the vast majority of modern drugs. Many small molecule compounds are non-immunogenic but immunoreactive haptens. As usual, they are conjugated to macromolecular antibodies as ADC payloads. , Some hormones and so on. Polypeptides are also an important part of modern drugs. For example, some common hormone drugs are polypeptides, and tumor neoantigen peptides have become a hot spot in drug development in recent years. The molecular weight of peptides is relatively large compared with that of compounds. Compared with antibodies, recombinant proteins or fusion proteins, their molecular weights are very small. They often lack more complicated secondary structure or spatial structure, etc., and their immunogenicity is weak. They are hapten-like substance. In theory, these hapten or hapten-like molecules alone cannot or are difficult to stimulate the body to produce antibodies, but after coupling the carrier antigen molecule and then immunizing the animal, antibodies that bind to the molecule itself can be prepared to construct a pair Its method for ELISA determination provides instrumental reagents.
These hapten molecules are very small and often have only one epitope, which cannot form a sandwich type that binds to the antibody at different sites; even if some hapten-like peptides may have two or more epitopes, the antibody is being prepared The reality is that it is often difficult to obtain antibodies that can form sandwich pairs for different epitopes. In this case, choosing to construct a competitive ELISA method is a strategy of tailoring measures according to your aptitude. Some molecules with large enough molecular weight and complex enough to contain multiple epitopes are fully antigenic molecules. For example, under the actual conditions that only one monoclonal antibody or only its polyclonal antibody can be used, it is also necessary to consider the selection and design competitiveness Of course, for the fully immune antibodies or protein molecules that have the conditions to be detected by the sandwich method, it is not unreasonable that the relevant researchers still consider using the competitive ELISA method for the determination.
How to comprehensively evaluate the experimental design of the target research based on the actual situation of antibody reagents available to me and the specific platform conditions of the laboratory, and successfully establish a “Fit-for-Purpose” analysis method based on the principle of competitive ELISA, which is also a pre-clinical and A specific and realistic technical problem in clinical PK or PK/PD evaluation research.
concentration usually have two basic Assay construction modes. One mode is to use monoclonal antibodies or polyclonal antibodies as solid-phase capture reagents, and the sample is free (in this article, free refers to non-solid phase). (Chemical) The drug and the labeled drug compete to bind to the solid-phase antibody, such as free drug and biotin-labeled drug competitively bind to the antibody coated in the well (see Figure 1). The other mode is to coat a certain concentration of drug on a solid-phase carrier, and use the free drug in the sample to compete with the solid-phase drug to bind free antibody (see Figure 2). In the application of antibody drug PK analysis, these two Assay modes can also be transformed into free drugs and labeled drugs competing for their solid-phase target molecules, or free drugs and solid-phase drugs competitively bind to labeled target molecules; for some Drugs can also be transformed into competitive binding to their receptor molecules. The specific design can be derived or transformed based on these two Assay modes.
Based on this model of competition between drugs and labeled drugs, drug molecules can be directly labeled with horseradish peroxidase (HRP) or alkaline phosphatase (AP). Such labeled drugs are mainly used for low sensitivity and based Competitive Assay development of traditional visible light absorption method detection platform. Based on the traditional light absorption detection platform, biotin labeling can also be used, and the high-sensitivity assay can be constructed with the help of streptavidin-biotin signal amplification system. With the development of molecular labeling technology and the continuous emergence of new platforms, if you need to develop more sensitive competitive Assays, you can also use fluorescein-labeled drug molecules to establish fluorescence detection methods, or you can use Eu (Europium ) Labeled drugs are detected based on a time-resolved fluorescence platform. If an analytical laboratory with an MSD platform can also use the company’s SULFO-TAG labeled drug molecules to develop electrochemiluminescence detection methods. Using the competitive model of labeled drugs and non-labeled drugs, the choice of labeled molecule and the type of signal produced can be based on the conditions of the laboratory platform or the detection function module possessed by the instrument and the actual sensitivity and labeling feasibility. Implement specific strategies.
When using the competitive model of free drugs and solid-phase drugs, one advantage is that it usually eliminates the trouble of molecular labeling and saves labeling costs, especially when the labeling is not feasible, for example, some drug molecules lack the base to bind the label. It is a good strategy to adopt this kind of competition method when the stability of the group or drug molecule is affected or other groups are affected. However, sometimes in practice we may find that the direct coating of the drug on the solid-phase carrier limits its effective binding to non-solid-phase antibodies, the overall signal of the Assay is not easy to increase, and the sensitivity is difficult to improve. It may be the result of immobilization. Its epitope is affected. However, if the unlabeled drug is directly solid-phased transformed into indirect solid-phase, the situation that this binding reaction is affected will sometimes be improved. For example, when the labeling is feasible, the drug should be biotin-labeled first to make the biotin The labeled drug is combined with Streptavidin coated on the solid-phase carrier to indirectly realize the solid-phaseization of the drug and then compete with the free drug (see Figure 3). Analysts can coat Streptavidin on their own, or they can purchase commercial coatings with Streptavidin materials and use them directly. Companies such as Thermo and MSD provide commercial Streptavidin Coated Plates.
The construction of analytical methodology model should be combined with specific laboratory platform conditions, available antibody conditions, the structure and properties of drug molecules, target research on the sensitivity and applicability of method detection range and other factors to formulate specific strategies.
One of the core principles of competitive Assays is “competition”. Therefore, the core point of establishing such methods is the composition of a competitive antigen-antibody reaction system, that is, to create an ELISA reaction system that can promote “competition”. The above two basic competitive ELISA Assay Formats are composed of three main components, namely: free drugs, labeled drugs or immobilized drugs, and antibodies. These three main components can be conceptualized as: competitive factor, competitive factor, and resource factor. These three factors are necessary to form a competitive environment that is a reaction system. The concentration of the labeled drug or the drug solid-phased in the carrier is fixed in the reaction system; the free drug concentration in the sample is not fixed, especially the standard curve is used in the PK quantitative analysis method, and the free drug and composition in the tested sample The free drugs with different concentration gradients of the standard curve are antibodies in the competitive binding system with the labeled drug or the drug solid-phased in the carrier. Therefore, the antibody in the reaction system can be said to be the “resource factor” in the competitive assay; The relationship between drugs and labeled drugs or drugs immobilized in the carrier is between competition and competition. For ease of explanation, in this article we refer to free drugs as “competitive factors” and labeled drugs or drugs immobilized in carriers as “Competed factor”, in fact, the two are mutually competitive factors.
The mutual influences or effects of some molecules in the micro world are in fact in common with the phenomena in the macro world or even in the human world. When certain resources are limited, humans will compete for the limited resources. That is to occupy resources competitively. Similarly, the basis of the competitive relationship between the competing factor and the competing factor in the competitive Assay reaction system is “limited resources”. For competitive ELISA, the antibody is limited or the total binding site of the drug contained in the antibody is limited. If the amount of antibody is too much or so-called saturation, the free drug cannot compete with the labeled drug or the immobilized drug, and such a competitive ELISA reaction system will fail.
Table 1 and Figure 4 show a competitive ELISA PK Assay case for a low-molecular-weight drug under development (Case 1). According to the dose and method of administration, it is predicted that an analysis method with a quantitative range of 0.391-25ng/mL needs to be developed. It adopts a mode in which solid phase drugs and free drugs competitively bind their monoclonal antibodies. For the standard curve prepared from the same batch, when the antibody concentration (shown as the dilution factor in actual use here) is very high (1/500), the signal values of the standards of different concentrations have no gradient changes and obvious differences. In this case Too high antibody concentration did not cause free drug competition to interfere with the binding of solid-phase drug and antibody and could not make the signal show a concentration gradient-dependent difference change. When other conditions are the same, reduce the monoclonal antibody dilution factor to 1:2000, and the difference in signal values of the standard products with different concentration gradients between the high concentration points of the standard curve will begin to appear, indicating that competition will occur; when you continue to reduce the monoclonal antibody dilution factor to 1: 5000, this concentration gradient-dependent signal difference is more significant, and the curve fitting trend is better.
For a competitive ELISA Assay, in addition to only providing limited “resource factors”, it is also necessary to consider the size of the power between the “competitive factor” and the “competed factor”, that is, the two competing objects cannot appear Competitive absolute power side. The absolute power will cause the corresponding competitors to have no ability to interfere with it because of their absolute advantage. For this, the phenomena of the human world and the phenomena of the microcosm are similarly connected. For example, two evenly matched people can stand in a stalemate for a long time. When one person fights two people at the same time, they may hold on for a while. When one person fights with more than a dozen people at the same time, because of the disparity in power, there is no ability to fight. It is extremely easy to be defeated by the opponent in an instant, and there is even no chance to resist. When the competitive ELISA method is established, if the proportion or concentration of the immobilized drug or labeled drug as the competitive factor is too high, the free drug as the “competitive factor” will not or hardly affect the binding of the competitive factor and antibody. Interference, the signal gradient between the different concentration standards of the standard curve cannot appear or is not significant, the standard curve is difficult to fit or the fitting quality is not high, and it does not have good measurement resolution for unknown samples, which affects the actual sample Real and accurate quantification. Therefore, when dealing with this problem in practice, attention should be paid to the proportional regulation of the competing factors participating in the reaction.
Observing the competitive ability of the competitive factor can be observed by normalizing the signal value of the sample to the binding percentage (B/B0). The interference of the competitive factor with the binding ability of the competitive factor and antibody is more intuitive. It is common in practice that only a gradient-dependent difference in binding percentage appears at a few high-concentration standard curve points, and B/B0 at low-concentration points has no significant difference or almost unchanged, that is, low-concentration standard products cannot participate well. Standard curve fitting. In this case, the competitive factor still occupies an absolute advantage over the low-concentration free drug, which affects the detection sensitivity of the method. You can try to lower the concentration or ratio of the competitive factor. As shown in Table 2 and Figure 5, Case 2: This method is a competitive ELISA method for antibody drugs. Assay Format is that free drugs compete with biotin-labeled drugs for their anti-idiotypic antibodies. When the dilution ratio of the prime-labeled drug is 1/30000, there is a concentration gradient-dependent difference in the binding percentage at several high-concentration standard curve points, but there is almost no difference in the standard B/B0 of low-concentration level. The accuracy of the low-concentration quality control product is also affected; when the dilution ratio of the biotin-labeled drug is 1/40,000, the standard B/B0 at the low-concentration level has a significant difference, and the standard curve is very good. Good fit, and the accuracy of low-concentration quality control products from the same preparation also tends to be better.
2: Comparison of method performance under different ratios of biotin-labeled drugs in Case 2
In social life, we often hear about unfair competition or unfair competition. Competitive ELISA Assay can also have equal competition and asymmetric competition. This article conceptualizes it as a balanced competition law and an unbalanced competition law. In the balanced competition method, the “competitive factor” and the “competitive factor” are activated simultaneously to the “resource factor” such as antibody binding reaction. In the competitive Assay Format of labeled drugs and non-labeled drugs, the labeled drugs and non-labeled drugs are synchronized Or it can be added to the solid-phase carrier coated with antibody to achieve a balanced competition with almost no time difference; in the competitive Assay Format of free drug and solid-phase drug, the sample containing free drug is first added to the solid-phase carrier coated with drug. In the phase carrier, antibodies are added to achieve a balanced competition. On the contrary, in the unbalanced competition law, the “competitive factor” and the “competitive factor” are activated asynchronously to the “resource factor” such as antibody binding reaction; in the competitive Assay Format of labeled drugs and non-labeled drugs, you can first Drugs or non-labeled drugs are added to the solid-phase carrier coated with antibodies, and after a period of reaction, another non-labeled drug or labeled drug is added to achieve non-equilibrium competition; in the competitive AssayFormat of free drugs and solid-phase drugs , First mix the free drug-containing sample with the antibody in a separate carrier for a period of time, and then add it to the drug-coated solid carrier, or first add the antibody to the drug-coated solid carrier. After reacting for a period of time, a sample containing free drug is added to it to participate in the reaction to achieve non-equilibrium competition. Generally speaking, researchers are accustomed to the operation process of the balanced competition law, but in some cases, the unbalanced competition law is adopted to optimize the performance of the Assay.
As shown in Case 3 in Table 3 and Figure 6, the solid-phase antigen and free antigen competitive binding antibody mode is used to quantitatively detect a certain peptide drug. In this case, the standard song and QC are also prepared from the same batch. Under the same other conditions, when the free drug and the antibody are mixed and reacted for a period of time and then added to the drug-coated microtiter plate, the performance of this analysis method is better than that of adding the free drug to the coated drug first It is much better to add antibodies to the microtiter plate immediately. In the unbalanced competition method, the B/B0 difference between the low concentration points of the standard curve is more significant, the standard curve fitting quality is better, the accuracy of QC is reliable, and the low concentration end of the standard curve is not only smooth in the unbalanced competition , QC from the same source also performed poorly.
To sum up, the competitive ELISA method for quantitative analysis of PK is a relatively difficult method. To make good use of this method in specific practice, the construction and reaction system should always be based on the fundamental principle of “competition”. optimization. Because the competition and interaction between molecules are behaviors in the micro world that cannot be seen by the naked eye, it is not a reference to radiate the phenomena of the macro world or the human world into the imagination and thinking of the relationships and functions between individuals in the micro world. Sexual thinking. Similar to the human world, no matter what kind of competitive analysis model is based on in practice, we must first create a competitive environment, that is, provide limited resources to trigger competition; we must also promote the generation of differentiated “competitive” effects, which is more important for competitiveness. In the ELISA method, competitive factors do not need to be evenly matched, but competitive factors with absolute advantages must be prevented from appearing. For ease of elaboration, the above content is mainly based on the traditional competitive ELISA detection mode to give examples and explanations. These practical strategies and key points in the article can be derived and optimized using some new platforms, and can also be extended to endogenous molecules. In the test, the author’s laboratory has specific practices of various derivative competitive ELISA methods, and the principles are the same, so I will not give examples one by one. In addition, the ELISA method used for PK quantification is the same as other commonly used PK quantification methods. After the method is established, various subsequent parameters need to be optimized and verified. There have been many related literatures, so this article will not repeat them, and grasp the proposal The strategy and key points will lay a good foundation for the smooth optimization and verification of subsequent parameters.
(Without permission, all pictures and data in the article must not be quoted)
Zhang Dengji, bachelor and Ph.D. respectively studied at Anhui Normal University, Shanghai Normal University & Shanghai Veterinary Institute of Chinese Academy of Agricultural Sciences and Fudan University. Their main focus is the analysis and evaluation of macromolecular drugs and cell gene therapy drugs. He used to work in the Macromolecular Bioanalysis Department of Shanghai WuXi AppTec for many years. After joining Medicilon, he created a biotechnology drug analysis team. Now he is the head of the drug bioanalysis department of Medicipua Biotechnology. He has more than 13 years of clinical and clinical experience. Former biotechnology drug analysis experience, responsible for the analysis and evaluation of multiple types of biopharmaceuticals related to Novartis, Pfizer, Lilly, J&J, Genentech, Medimmune, Takeda, CSPC and other pharmaceutical companies. He has a wealth of theoretical foundation and practical experience in the analysis of pharmacokinetics, toxicokinetics, immunogenicity and biomarkers of biotechnology drugs. He has written many related articles and applied for inventions in relevant countries as the first inventor. Of the five patents, three have been authorized.
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