Experimental methods and classification of immunofluorescence technology

　　1. Fluorescence immunoassay

　　The principle is to use a sandwich method of a pair of monoclonal antibodies. The substrate is 4-methylumbelliferyl phosphate, and the fluorescence emitted by the product is detected . The fluorescence intensity is proportional to the Mb concentration, and the result can be obtained within 8 minutes. The result is expressed as the rate of Mb release per hour (△Mb). This method has good repeatability, a wide linear range, and is rapid, sensitive, and accurate.

　　Taking the double antibody sandwich method as an example, firstly, the specific antibody is connected to the solid phase carrier to form a solid phase antibody. Unbound antibodies are removed, and then the specimen to be tested is added so that the protein antigen in it forms an antigen-antibody complex with the solid phase antibody. Unbound substances are washed away, and then fluorescently labeled antibodies are added to specifically bind to the antigen to form an antibody-antigen-antibody complex. Finally, the protein antigen can be quantified according to the fluorescence intensity.

　　Traditional fluorescence immunoassay is greatly affected by background fluorescence. Time-resolved fluorescence immunoassay uses rare earth metals with extremely long lifespans, such as europium, as markers. After adding normal fluid, it is excited and measured, which can effectively remove the interference of short-life background fluorescence.

　　2. Radioimmunoassay

　　Radioimmunoassay is a method in which excess unlabeled antigen and antigen labeled with radioactive substances are competitively combined with antibodies to form radioactive antigen-antibody complexes and non-radioactive antigen-antibody complexes, as well as excess labeled antigen and unlabeled antigen. Then, by centrifugal precipitation and other methods, the antigen-antibody complex is separated from the free antigen, and the radioactivity intensity is measured and compared with the standard curve, so that the unlabeled antigen to be tested can be quantified.

　　RIA has high sensitivity and specificity in measuring serum protein, and can accurately quantify to the ng/ml level. However, the early methods were cumbersome to operate, time-consuming, and had radioactive contamination. In recent years, with the application of monoclonal antibodies, the sensitivity of RIA has been greatly improved, and the operation has been greatly simplified. Commercial kits are now available and easy to use.

　　3. Enzyme-linked immunosorbent assay (ELISA)

　　There are two types of ELISA methods: competitive method and sandwich method. The competitive method is based on the principle that standard or serum Mb and Mb coated on microplates competitively bind to monoclonal antibodies. The minimum detection limit of this method is 10μg/L, and the linear range is 1 000ug/L. The sandwich ELISA method has a good correlation with EIA (r=0.92). The ELISA method has the advantages of high sensitivity, strong specificity, good precision, simple operation, suitable for the detection of multiple specimens, no need for special instruments and equipment, and is easy to promote and popularize. However, it is not suitable for rapid detection in emergency cases.

　　Take the double antibody method as an example. First, the antigen is coated, and then the primary antibody is added to form an antigen-antibody complex with the coated antigen. Then, the enzyme-labeled secondary antibody is added to form an antigen-antibody-antibody complex. Finally, the substrate is added, and the enzyme catalyzes the substrate to generate a product. The protein antigen can be quantified by the amount of product generated.

　　4. ELISA with biotin-avidin system

　　By utilizing the property that one avidin molecule can bind to four biotin molecules, the sensitivity of the traditional ELISA with higher sensitivity is significantly amplified.

　　5. Time-resolved fluorescence immunoassay

　　Time resolved fluorescence immunoassay (TRFIA) is a non-isotopic immunoassay technique that uses lanthanide elements to label antigens or antibodies. Based on the luminescence characteristics of lanthanide chelates, it uses time-resolved technology to measure fluorescence and simultaneously detects two parameters, wavelength and time, for signal resolution. This can effectively eliminate the interference of non-specific fluorescence and greatly improve the sensitivity of analysis.

　　6. Dissociation Enhanced Lanthanide Fluorescence Immunoassay

　　Dissociation Enhanced Lanthanide Fluor Immunoassay (DELFIA) is a type of time-resolved fluorescence immunoassay. It uses a chelating agent with a bifunctional group structure to connect one end to europium (Eu) and the other end to the free amino group on the antibody/antigen molecule to form an EU-labeled antibody/antigen, which generates an immune complex after an immune reaction. Since the fluorescence intensity of this complex in water is very weak, an enhancer is added to dissociate Eu3 from the complex, and the free Eu3 chelates with another chelating agent in the enhancer to form a colloidal molecular cluster. This molecular cluster can emit strong fluorescence under the excitation of ultraviolet light, and the signal is enhanced by a million times. Because this analytical method uses a dissociation enhancement step, it is called dissociation enhanced lanthanide fluorescence immunoassay. [page]

　　2. Fluorescent antibody staining methods and their classification

　　1. Direct staining method

　　The labeled specific fluorescent antibody is directly added to the antigen sample, stained at a certain temperature and time, and the excess fluorescent antibody that does not participate in the reaction is washed away. Under a fluorescent microscope , the fluorescence emitted by the specific binding product formed by the antigen being tested and the fluorescent antibody can be seen. The advantages of the direct staining method are: high specificity, simple operation, and relatively fast. The disadvantages are: one labeled antibody can only detect one antigen, and the sensitivity is poor. The direct method should set up negative and positive sample controls and inhibition test controls.

　　2. Indirect staining method

　　If an unknown antigen is to be examined, a known unlabeled specific antibody (first antibody) is first used to react with the antigen specimen. After a certain period of time, the unreacted antibody is washed away, and then a labeled anti-antibody, i.e., antiglobulin antibody (second antibody), is used to react with the antigen specimen. If the antigen and antibody in the first step react with each other, the antibody is fixed or combined with the anti-antibody labeled with fluorescein to form an antigen-antibody-antibody complex. The unreacted labeled anti-antibody is then washed away, and fluorescence can be seen under a fluorescence microscope. In the indirect staining method, the unlabeled antibody used in the first step plays a dual role, acting as an antibody for the antigen and as an antigen for the anti-antibody in the second step. If an unknown antibody is to be examined, the antigen specimen is a known serum to be examined as the first antibody, and the other steps are the same as for the antigen.

　　Since immunoglobulins are species-specific, labeled antiglobulin antibodies must be prepared by immunizing other animals with serum globulins of the same species as the primary antibody.

　　The advantage of the indirect staining method is that it can detect both unknown antigens and unknown antibodies; a labeled antiglobulin antibody can bind to the antibodies of all animals of the same species to detect various unknown antigens or antibodies with high sensitivity. The disadvantages are: due to the large number of factors involved in the reaction, the possibility of interference is also large, and the judgment result is sometimes difficult, the operation is cumbersome, there are many controls, and the time is long. The indirect method should set up negative and positive sample controls, and should also set up an intermediate layer control (that is, the intermediate layer plus negative serum instead of positive serum).

　　3. Anti-complement staining

　　The anti-complement staining method is referred to as the complement method. It is a modified method of the indirect staining method and was first established by Goldwasser et al. This method uses the principle of complement binding reaction to label anti-complement antibodies with fluorescein to identify unknown antigens or unknown antibodies (serum to be tested). The staining procedure is also divided into two steps: first, unlabeled antibodies and complement are added to the antigen specimen to react, washed with water, and then labeled anti-complement antibodies are added. If the antigen and antibody react to form a complex in the first step, the complement will be bound by the antigen-antibody complex. In the second step, the fluorescein-labeled anti-complement antibody added will react specifically with the complement to form an antigen-antibody-complement-anti-complement antibody complex, which emits fluorescence.

　　The anti-complement staining method has the same advantages as the indirect method. In addition, it has its own unique advantages: only one labeled anti-complement antibody is needed to detect various antigen-antibody systems. Because complement has no specificity, it can react with any mammalian antigen-antibody system. Its disadvantages are that there are many components involved in the reaction, and the staining procedure is complicated and troublesome.

　　In addition to the above three methods, there are some methods evolved on this basis, such as double-layer method, sandwich method, mixed method, three-layer method, antibody-anti-complement method and so on.