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Analysis of the role of affinity determination in the development of biological drugs

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Drugs exert various pharmacological functions in vivo and in vitro by binding to the target, that is, interacting.Affinity (affinity) is a characteristic function of the relative state between candidate molecules, target molecular epitopes and candidate molecule-target molecule conjugates during the reversible reaction process, and can evaluate the strength of the binding between candidate molecules and target molecules. For biopharmaceutical development, affinity is one of the key functional attributes for judging drug efficacy. Accurate evaluation of drug candidate affinity is the first step to understand the mechanism of action of drug candidates.

As an index to evaluate the quality of drugs

The combination of drugs and target molecules can be divided into irreversible binding and reversible binding. Among the listed drugs, most drugs and target molecules are reversibly combined, which is conducive to easier metabolism of drugs after entering the human body, reducing the possibility of existence Of long-term accumulation and safety risks. In the development of biopharmaceuticals, ELISA technology is used for affinity determination to analyze the quality of drugs. For example, researchers have expressed the G protein of rabies virus (RV) CTN-1 strain using a prokaryotic expression system, and evaluated its basic functions [1].

The researchers used RT-PCR to amplify the G protein gene of the RV CTN-1 strain, inserted the prokaryotic expression vector pGEX-5X-1, constructed the recombinant expression plasmid pGEX-5X-1-CTN, transformed it into Rosetta(DE3)pLysS, and induced it by IPTG expression. After the expressed recombinant protein was purified by affinity chromatography, the indirect ELISA method was used to detect its binding performance and affinity with positive serum. The study successfully used prokaryotic expression and purification to obtain the RV CTN-1 strain G protein with high purity, good specificity, strong affinity, and high sensitivity. It was used to study the epitope of the G protein and establish a target for the RV G protein. The ELISA antibody detection method and the sorting and detection of neutralizing antibodies laid the foundation.


Screen antibodies

Antibodies are immunoglobulins that are naturally produced to help the immune system resist internal and external invasions of cells. Antibody affinity refers to the binding force between antibodies and antigenic determinants, and its size is a very important indicator for evaluating antibody effects. Determining the affinity of antibodies to specific antigens is very important for screening antibodies. If researchers use antibody library technology to screen, clone, and express single-chain antibodies against asialoglycoprotein receptors in vitro, and improve the screening of single-chain antibodies, the positive rate of screening is further improved, and the obtained The affinity determination of single-chain antibodies makes it useful as a targeted molecule for liver cancer gene therapy and lays the foundation for its application as a gene therapy for liver cancer in vivo.

In the research and development of biopharmaceuticals, the determination of antibody affinity is of great significance, such as monoclonal antibodies. The determination of antibody affinity can provide a basis for selecting monoclonal antibodies for different purposes. It can also pre-select monoclonal antibodies early after successful hybridoma fusion, which has achieved savings. Materials and the purpose of shortening the experiment cycle. Medicilon provides biopharmaceutical research and development services and has extensive experience in molecular biology, cell biology, in vitro biology and structural biology. From the initial cDNA library construction to drug design, through protein purification, structure determination and analysis, we provide a complete set of biological services.

Generally speaking, the higher the affinity constant K, the stronger the ability of the antibody to bind to the hapten. However, it does not mean that the stronger the affinity, the stronger the effect. In many cases, the appropriate affinity or even the kinetic parameters related to the appropriate affinity should be selected according to the mechanism of drug action. Dissociation rate constant (Koff) and binding rate constant (Kon). For example, in the process of solid tumor treatment with antibody drugs, too strong affinity antibodies may cause macromolecular antibodies to only accumulate near the blood vessels of the solid tumor, thereby affecting the diffusion and accumulation of antibodies in the solid tumor site, resulting in less drug effect At this time, antibodies with weaker affinity may have better efficacy, and larger Koff parameters will have better effects.

Affinity testing is not only more important in drug development, but also has a greater role in clinical applications. In recent years, many scholars have used antibody affinity tests to identify the primary infection and past infections of pathogens, to solve the problem of atypical symptoms of related diseases due to immunity and other reasons, and to provide scientific basis for formulating preventive and therapeutic measures. For example, the antibody affinity test is one of the best ways to determine the primary and non-primary infections of infectious diseases, and it is an important supplement to traditional etiology and immunological detection techniques.

[1] Prokaryotic expression and basic function evaluation of G protein of rabies virus CTN-1 strain [J].

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