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Immunoassay is a method of detecting poisons by competitive binding antibodies between poisons and labeled poisons. It can be used in screening tests of some toxic drugs.The immunoassay method is used for detection. When no unlabeled toxic drug is added, the antibody will completely combine with the labeled toxic drug to form a labeled toxic drug antibody complex.After the addition of unlabeled toxic drugs, unlabeled toxic drugs will also bind with antibodies to generate unlabeled toxic drugs antibody complexes, thus inhibiting the binding reaction between labeled toxic drugs and antibodies and reducing the content of labeled toxic drugs in the generated products.If the amount of antibody and labeled drug is fixed, there is a functional relationship between the amount of unlabeled drug added and the amount of labeled drug in the complex.Select an appropriate method to detect the labeled toxic drugs in the complex, and then the amount of toxic drugs in the test material can be calculated accordingly.[1]
In drug analysis, the application of immunoassay mainly focuses on the following aspects: (1) In experimentsPharmacokineticsAnd determination in clinical pharmacyBioavailabilityandPharmacokineticsParameters, etcBiopharmaceuticsTo understand the absorption, decomposition, metabolism and excretion of drugs in the body;(2) In the clinical testing of drugsTreatment indexSmall or more than the safe dose is prone to serious adverse reactions or the optimal treatment concentration andToxic reactionThe blood concentration of drugs with cross concentration shall be monitored;(3) From fermentation broth orcell cultureThe content of effective components in the liquid is quickly measured to realize online monitoring of the production process;(4) Evaluate whether there are specific trace harmful impurities in the drug.
Basic principle: soluble antigen and corresponding antibody contact each other in solution or gel to form insoluble antigen antibody complex precipitation.
Single immunodiffusion
Basic principle: only one of the two components, antigen and antibody, is diffused, and the other is fixed in the gel.
Double immunodiffusion
Basic principle:Soluble antigenWith the corresponding antibody in theagarIn mediaInterdiffusionAnd form a certain type of specific precipitation line after meeting each other.
Electrophoretic technique
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Basic principle: combination of immunodiffusion and electrophoresis.
Basic principle: Most protein antigens are negatively charged in alkaline buffer and move from negative electrode to positive electrode during electrophoresis.The antibody only has a weak negative charge in alkaline buffer, andrelative molecular massLarge, less electrophoretic forceagarElectroosmosisMove from positive pole to negative pole under the action of force.Results Antigen and antibody directed convection reacted when the two holes met, and formed a white precipitation line visible to the naked eye at a proper proportion.
Rocket immunoelectrophoresis
（Rocket immunoelectrophoresis）
Basic principle: also known as immune diffusion, the antigen can be detected in theagaroseWhen the ratio of the two is appropriate, a conical precipitation peak will be generated in a short time.In a certain concentration range, the height of the precipitation peak is proportional to the antigen content.
Immunoelectrophoresis
Basic principle: first make the sample to be sideagarGel electrophoresis, each protein antigen component is divided into different zones, and then dig a small groove parallel to the electrophoresis direction, add the correspondingantiserumThe protein antigen components divided into zones are subject to bidirectional immunodiffusion to form a precipitation arc at the corresponding positions of each zone.
Basic principle: according toAntigen antibodyThe principle of specific bindingradio isotopeLabel antigen or antibody, and determine the amount of antibody or antigen in the sample to be tested qualitatively or quantitatively according to the amount of radiation.
Rationale: ByMolecular targetBinding method, the nanometer guide magnet beads（Immune magnetic bead）It is bound to the protein antibody to be tested and solidified inGmr （GMR, Giant Magneto Resistance) chip surface, GMR chip basedGiant magnetoresistance effectThe immune magnetic beads on the chip surface will strongly affect the original resistance of GMR, and quantitative determination of antibody content in samples can be realized according to the change rate of GMR resistance.Magnetic sensitive immunoassayTechnology belongs to the family of biochip technology.
Fluorescence immunoassay (FIA)
Basic principle:fluoresceinLabelled antibodyOr antigen astracerThe principle of ELISA is similar to that of ELISA.This method can not only quantify the antigen and antibody in liquid, but also qualitatively and quantitatively determine the antigen and antibody in tissue sections.Generally due to theAutofluorescenceAnd excitationLight scatteringThe background fluorescence is high, which affects the sensitivity of the determination.GenerallyLanthanideAs a fluorescent marker (tracer).After the tracer is combined with the corresponding antigen or antibody, the fluorescence detector is used to checkFluorescence phenomenonOr measurementfluorescence intensitySo as to judge the existence, location and distribution of antigen or antibody or detect the content of antigen or antibody in the tested sample.
Colloidal gold immunoassay (CGIA)
Basic principle: colloidal gold is used as a tracer marker, which mainly takes advantage of the high electron density of gold particles. Dark brown particles can be seen under the microscope at the gold labeled protein junction. When these markers gather in large quantities at the corresponding ligand, red or pink spots can be seen with the naked eye.
Chemiluminescence immunoassay (CLIA)
Basic principle: use chemical or bioluminescence system asAntigen antibody reactionThe luminous substances can be directly used asAntigen antibodyIt can also be used in free form in the luminescence reaction of antigen or antibody labeled by catalyst (enzyme) and adjuvant.
