Immunoassay is a method of detecting toxicants by using the toxicants and labeled toxicants to competitively bind antibodies. Can be used for screening tests for certain poisons. The immunoassay is used for detection. When no unlabeled poison is added, the antibody is completely combined with the labeled poison to form a labeled poison-antibody complex. After adding the unlabeled poisonous drug, the unlabeled poisonous drug will also combine with the antibody to form the unlabeled poisonous drug-antibody complex, thereby inhibiting the binding reaction of the labeled poisonous drug and the antibody, and reducing the content of the labeled poisonous drug in the generated product. If the amount of the antibody and the labeled poison is fixed, there is a certain functional relationship between the amount of the unlabeled poison added and the content of the labeled poison in the complex. Select a suitable method to detect the labeled poison in the complex, and then calculate the amount of the poison in the test material.
Application
In drug analysis, the application of immunoassay mainly focuses on the following aspects:
(1) Determination of important data in biopharmaceuticals such as bioavailability and pharmacokinetic parameters in experimental pharmacokinetics and clinical pharmacology, in order to understand the absorption, decomposition, metabolism and excretion of drugs in the body;
(2) In the clinical testing of drugs, monitor the blood concentration of drugs with a small therapeutic index, serious adverse reactions that exceed safe doses, or overlap between optimal therapeutic concentrations and toxic reaction concentrations;
(3) Rapidly measure the content of effective components from fermentation broth or cell culture broth in drug production to realize on-line monitoring of the production process;
(4) Evaluate whether there are specific trace harmful impurities in the drug.
Classification
Label-free immunoassay techniques: immunodiffusion, immunoelectrophoresis
Labeled immunoassay techniques: enzyme immunoassay, radioimmunoassay, magnetic sensitivity immunoassay, other immunoassays (fluorescence immunoassay, colloidal gold immunoassay, luminescence immunoassay, and ferritin immunoassay, etc.)
immune diffusion
Rationale: Soluble antigens and corresponding antibodies contact each other in solution or gel to form insoluble antigen-antibody complex precipitates.
Single immunodiffusion
Basic principle: It means that only one of the two components, antigen and antibody, diffuses, and the other is immobilized in the gel.
Double immunodiffusion
Basic principle: refers to the mutual diffusion of soluble antigen and corresponding antibody in agar medium, and a certain type of specific precipitation line is formed after meeting each other.
electrophoresis
Basic principle: the combination of immunodiffusion and electrophoresis.
Type: Convective immunoelectrophoresis, rocket immunoelectrophoresis, immunoelectrophoresis, two-dimensional immunoelectrophoresis (cross-immunoelectrophoresis)
counterimmunoelectrophoresis
Basic principle: Most protein antigens are negatively charged in alkaline buffers and move from negative to positive during electrophoresis. Antibodies only have a weak negative charge in alkaline buffers, and their relative molecular mass is relatively large, and their electrophoretic force is small. As a result, the antigen and antibody are directional convection, react when they meet between the two wells, and form a visible white precipitation line at the right ratio.
Rocket immunoelectrophoresis
Basic principle: Also known as immunodiffusion, the antigen moves in agarose containing quantitative antibodies. When the ratio of the two is appropriate, a conical precipitation peak is generated in a short time. Within a certain concentration range, the height of the precipitation peak is proportional to the antigen content.
Immunoelectrophoresis
Basic principle: first perform agarose gel electrophoresis on the sample to be side, each protein antigen component is divided into different zones, and then dig a small groove parallel to the direction of electrophoresis, add the corresponding antiserum, and divide the protein antigen components into zones. For two-way immunodiffusion, precipitation arcs were formed at the corresponding positions of each zone.
Marking Technology
Basic principle: Use tracer substances such as fluorescein, isotopes or enzymes to label antibodies (or antigens) for antigen-antibody reaction, and monitor the immune response by measuring the markers in the immune complex.
The main types of labeling immunotechnology: radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, chemiluminescence immunoassay
Radioimmunoassay (RIA)
Basic principle: According to the principle of antigen-antibody specific binding, the antigen or antibody is labeled with radioisotope, and the amount of antibody or antigen in the specimen to be tested is qualitatively or quantitatively determined according to the amount of radiation.
Enzyme Immunolabeling Technology (ELISA)
Basic principle: It refers to the antigen-antibody reaction of enzyme-labeled antibody or enzyme-labeled antibody, and then produces a color reaction with the substrate through the enzyme for quantitative determination.
Magnetic Sensitivity Immunoassay (MI)
Basic principle: Through the molecular target binding method, the nano-scale conductive magnetic beads (immunomagnetic beads) are combined with the protein antibody to be tested and cured on the surface of the giant magnetoresistance (GMR, Giant Magneto Resistance) chip. GMR chip-based giant magnetoresistance The immunomagnetic beads on the chip surface will greatly affect the original resistance of GMR, and the content of the antibody to be tested in the sample can be quantitatively determined according to the rate of change of the GMR resistance. Magnetic immunoassay technology is a member of the biochip technology family.
Fluorescent Immunoassay (FIA)
Basic principle: a new immunoassay technology using fluorescein-labeled antibodies or antigens as tracers, the principle is similar to ELISA. This method can not only quantify antigens and antibodies in liquids, but also qualitatively and quantitatively identify antigens and antibodies in tissue sections. Generally, due to the autofluorescence of samples and reagents and the scattering of excitation light, the background fluorescence is high, which affects the sensitivity of the assay. Lanthanides are generally used as fluorescent markers (tracers). After the tracer is combined with the corresponding antigen or antibody, the fluorescence phenomenon is observed or the fluorescence intensity is measured with the help of a fluorescence detector, so as to judge the existence, localization and distribution of the antigen or antibody or detect the content of the antigen or antibody in the tested sample.
Colloidal Gold Immunoassay (CGIA)
Basic principle: Colloidal gold is used as a tracer marker, which mainly utilizes the characteristics of gold particles with high electron density. At the binding site of gold-labeled protein, dark brown particles can be seen under the microscope. When these markers are in large quantities at the corresponding ligands When aggregated, red or pink spots are visible to the naked eye.
Chemiluminescence Immunoassay (CLIA)
Basic principle: Using chemical or bioluminescence system as the indicator system of antigen-antibody reaction, to quantitatively detect antigen or antibody, luminescent substance can be directly used as antigen-antibody marker, or can be used as catalyst (enzyme) and auxiliary agent in free form in the luminescence reaction of the labeled antigen or antibody.