Immunogenicity Testing Method of Biotech Drugs

Immunogenicity Testing

Immunogenicity refers to the nature of drugs that stimulate the body to form specific antibodies or sensitized lymphocytes. In the development of biotechnology drugs, immunogenicity testing is an important task. For patients, immunogenicity will affect the safety and effectiveness of drugs, and even cause life-threatening dangers to patients because of the crossover of anti-drug antibodies and endogenous proteins. For companies, immunogenicity testing is not good. Only discovered the problem of anti-drug antibodies will increase the risk and loss of enterprise R&D. So what are the commonly used immunogenicity testing methods?

ELISA-bridge method

The immunogenicity test of antibody drugs is an important part of the application for clinical trials and registration of biotechnology drugs. Although existing animal tests have shown that immunogenicity does not necessarily produce the expected immune response in the human body, the evaluation of the drug immune response Still very important. The process of fixing the antigen or antibody is called coating. In other words, coating is the process of binding the antigen or antibody to the surface of the solid carrier. The ELISA-bridge method coats the drugs and detects them with labeled drugs. The advantage of this method is that it can detect various antibody subtypes, without species specificity, and can be tested with high throughput. The disadvantage is that it is not easy to detect antibodies with low affinity, and the epitope of the drug may be masked or changed during coating or labeling. , Susceptible to interference from the drug itself. Medicilon provides immunogenicity testing services, mainly using mice, rats, guinea pigs, rabbits, dogs, and non-human primates for immunogenic toxicity tests.

ELISA-direct method

Antibodies are generally coated by direct adsorption, and most protein antigens can also be coated by methods similar to antibodies. ELISA-direct method to coat the drug and detect with labeled antibody. The advantage of ELISA-direct method for immunogenicity detection is that it may increase the ability to detect low-affinity antibodies and has high throughput. However, when the antigenic determinant exists in or adjacent to the hydrophobic region, the direct adsorption of the antigen and the solid-phase carrier can make the antigenic determinant not fully exposed. In this case, the direct coating effect is not good, and indirect capture package can be used. By law. In addition, the epitope of the drug may be masked or changed when directly coated, only a single subtype is detected, which is species-specific, and low-affinity antibodies are lost during multiple washings. The reagents may be different between the reference product and the sample.

ELISA-indirect method

ELISA-indirect method to coat monoclonal antibodies or biotin, and then add drugs, that is, the specific antibody against the antigen is pre-coated, and then the antigen is solid-phased through the antigen-antibody reaction. The antigen indirectly bound to the solid phase is far away from the surface of the carrier, and its antigenic determinants are also fully exposed. The indirectly coated antigen is subjected to the affinity chromatography of the solid phase antibody, and the purity of the antigen coated on the solid phase is greatly improved. Therefore, the antigen with more impurities can also be captured and coated to test the specificity and sensitivity This can be improved, and the repeatability is also good. Another advantage of indirect coating is that the amount of antigen is less, which is only 1/10 or even /100 of that of direct coating. Non-protein antigens that are not easily adsorbed on the polystyrene carrier can be coated in a special way.

Radioimmunoassay (RIA)

RIA is a microanalysis method that uses a radionuclide labeling antigen or antibody, and then combines with the tested antibody or antigen to form an antigen-antibody complex for analysis. It has both the sensitivity of radioisotopes and the specificity of antigen and antibody reactions. The method also has the advantages of high accuracy and precision, easy standardization, simple operation and economy. Because RIA has the advantages of high sensitivity, strong specificity, simple measurement and low cost, RIA has certain vitality in application. However, because its most fatal weakness is the use of radionuclides, and the effective use time of markers is short, it is difficult to realize the automation of operation and measurement, and its further development is subject to some limitations. Now immunoassay technologies are developing towards the direction of non-isotopic labeling immunoassay.

Electrochemiluminescence (ECL)

Electrochemiluminescence (ECL) is derived from electrochemical methods and chemiluminescence methods. It can not only be applied to all immunoassays, but also can be used for DNA/RNA probe detection. Its advantages are the liquid phase method, which can detect various antibody subtypes, has no species specificity, high throughput, can use high concentration matrix, large detection surface area and stable signal. The disadvantage is that two kinds of markers (biotin and TAG) need to be prepared, the epitope of the marker molecule may change or the labeling process will change the molecule, and the reagents used are not universal.

Surface plasmon resonance

The advantage of the surface plasmon resonance method is the liquid phase method, which does not require a conjugate (enzyme-labeled antibody), and can detect antibodies with different affinities and antibodies of various subtypes, without species specificity. The disadvantage is that chemical linkage may affect the molecule, and the linkage with dextran may affect the exposure of the epitope. Refolding may degrade the molecule. The reagents used are poor in versatility and low throughput. If the antibody produced is of the same subtype of the drug, It is difficult to confirm the anti-antibody reaction of a humanized antibody and the sensitivity is low.

Enzyme-linked immunospot method (ELISpot)

The principle of enzyme-linked immunospot (ELISpot) is similar to that of ELISA. It is also a method for detecting cytokines or other soluble proteins produced by cells. It can not only determine the amount of cytokines, but also count the frequency and sensitivity of cells secreting this cytokine. It is higher than ELISA, and the capture antibody used in the experiment will not affect the secretion of cytokines by activated cells. The disadvantage is that it is more complicated and time-consuming than the ELISA technology, and the experimental conditions need to be strictly controlled. The operator needs to be skilled and able to analyze the experimental results to reduce experimental deviation; it is a semi-quantitative method.

Immuno-PCR method (IPCR)

The immuno-PCR method (IPCR) is established on the basis of ELISA, which uses PCR amplification to replace the enzyme-catalyzed substrate of ELISA for color development. PCR has a strong amplification ability, can quantitatively detect DNA and RNA, and has very high sensitivity and specificity. Therefore, the specific antibody that binds to the antigen is combined with the DNA through the linking molecule, and then the antigen is quantitatively detected by PCR amplification. The sensitivity of IPCR is higher than that of ELISA. Compared with the corresponding ELISA, IPCR can increase the sensitivity of antibody detection by at least 1000 times, and only the dilution method in this method can eliminate the interference of the matrix in the biological sample.

The currently reported IPCR all use the antigen to be tested to directly adsorb the solid phase, so the homogeneity of the solid phase has a great influence on the results; at the same time, other components in the tested sample can also be adsorbed to the solid phase, which is easy to cause excessive background or accuracy. Degree drops. Some antigens that are difficult to adsorb to the solid phase cannot be detected by immuno-PCR. The specificity and homogeneity of the linking molecule have a great impact on PCR, and the PCR amplification process is relatively simple. For example, a microplate is used as a solid phase and a matching PCR instrument is required. , Otherwise it needs to be moved into the reaction tube, which will inevitably lead to a large error, and magnification can produce significant differences. IPCR has broad application prospects, but it is necessary to further improve the experimental process of IPCR and the development of supporting reagents.

The immunogenicity test of antibody drugs is not only a problem of drug treatment effect, but also a problem of drug safety. The side effects of people’s anti-antibody is still unclear. However, the impact of anti-antibody production on the drug itself and the potential allergic reaction cannot be ignored. Therefore, the immunogenicity test of antibody drugs needs continuous research.

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