Experimental methods and classification of immunofluorescence technology

Experimental methods and classification of immunofluorescence technology

I. Immunolabeling and its classification

Fluorescent immunoassay

The principle is a sandwich method using a pair of monoclonal antibodies . The substrate was phosphate--4-methylumbelliferone, and the fluorescence emitted by the product was detected. The fluorescence intensity was proportional to the concentration of Mb, and the result was obtained within 8 min. The results are expressed as the rate of release of Mb per hour (ΔMb). The method has good repeatability, wide linear range, and is fast, sensitive and accurate.

Taking the double-antibody sandwich method as an example, a specific antibody is first linked to a solid phase carrier to form a solid phase antibody. The unbound antibody is removed, and then the test sample is added to form a antigen-antibody complex with the solid phase antibody. The unbound material is removed by washing, followed by the addition of a fluorescently labeled antibody to specifically bind to the antigen to form an antibody-antigen-antibody complex. Finally, protein antigens can be quantified based on fluorescence intensity.

The traditional fluorescence immunoassay is greatly interfered by the background fluorescence. The time-resolved fluorescence immunoassay is a rare earth metal with a long life span, such as strontium, as a marker. After the addition of normal liquid, the excitation assay can effectively remove the short-lived background fluorescence. Interference.

2. Radioimmunoassay

Radioimmunoassay is an antigen that is labeled with an excess of unlabeled antigen and a radioactive substance, competitively binds to an antibody to form a radioactive antigen-antibody complex and a non-radioactive antigen-antibody complex, and has excess labeled antigen and Unlabeled antigen. Then, the antigen-antibody complex is separated from the free antigen by centrifugation and the like, and the radioactivity intensity is compared with a standard curve to quantify the unlabeled antigen to be tested.

The RIA method has high sensitivity and specificity for the determination of serum protein, and can be accurately quantified to the ng/ml level. But the early methods were cumbersome to operate, time consuming, and radioactive. In recent years, with the application of monoclonal antibodies, the sensitivity of RIA has been greatly improved, and the operation has been greatly simplified, and commercial kits have been supplied, which is convenient to use.

3. Enzyme-linked immunosorbent assay (ELISA)

There are two types of ELISA methods: competition method and sandwich method. The competition method is based on the principle that standard or serum Mb and coated Mb on microplates are competitively combined with monoclonal antibodies. The minimum detection limit is 10 μg/L and the linear range is 1 000 ug/L. . The sandwich ELISA method has a good correlation with EIA (r=0.92). The ELISA method has high sensitivity, strong specificity, good precision and simple operation. It is suitable for the detection of multiple specimens, and does not require special equipment and equipment, and is easy to popularize. But it is not suitable for rapid detection of emergency.

Take the double-antibody method as an example. The antigen is first coated, and then the primary antibody is added to form an antigen-antibody complex with the coated antigen. The enzyme-labeled secondary antibody is then added to form an antigen-antibody-antibody complex. Finally, the substrate is added and the product is produced by the enzyme catalyzed substrate. Protein antigens can be quantified by the amount of product produced.

4. Enzyme-free method for coupling biotin-avidin system

By utilizing the characteristics that one avidin molecule can bind to four biotin molecules, the sensitivity of the conventional sensitivity-sensitive enzyme-free method has a significant amplification effect.

5. Time-resolved fluorescence immunoassay

Time resolved Fluor immunoassay (TRFIA) is a non-isotopic immunoassay technique that uses a lanthanide to label an antigen or antibody and measure the fluorescence using time-resolved techniques based on the luminescent properties of the lanthanide chelate. Simultaneous detection of wavelength and time parameters for signal resolution 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 one of the time-resolved fluorescence immunoassays. It uses a chelating agent with a bifunctional group structure to link one end to Eu (Eu) and the other end to a free amino group on the antibody/antigen molecule to form an EU-labeled antibody/antigen, which is immunologically complexed to generate an immune complex. Object. Since the fluorescence intensity of this complex in water is very weak, an enhancer is added to dissociate Eu3 from the complex, and free Eu3 is chelated with another chelating agent in the enhancer to form a colloidal molecule. This group of molecules can emit strong fluorescence under the excitation of ultraviolet light, and the signal is enhanced by a million times. Because this analytical method uses an understanding of the enhancement step, it is called dissociation-enhanced lanthanide fluorescence immunoassay.

Second, fluorescent antibody staining methods and their classification

Direct staining

The labeled specific fluorescent antibody is directly added to the antigen sample, and after being stained at a certain temperature and time, the excess fluorescent antibody not participating in the reaction is washed away, and the specific combination formed by the test antigen and the fluorescent antibody can be seen under the fluorescence microscope. And the fluorescence emitted. The advantages of the direct staining method are: high specificity, easy operation, and relatively fast. The disadvantage is that a labeled antibody can only examine one antigen and is less sensitive. The direct method should be set up for yin and positive specimens, and the control test should be suppressed.

2. Indirect staining

If an unknown antigen is detected, first react with an antigen sample using a known unlabeled specific antibody (primary antibody), and after a certain period of time, wash away the unreacted antibody, and then use the labeled anti-antibody, anti-globulin antibody (p. The second antibody) reacts with the antigen sample. If the antigen antibodies in the first step react with each other, the antibody is immobilized or bound to the fluorescein-labeled anti-antibody to form an antigen-antibody-antibody complex, and then washed away without reaction. The labeled anti-antibody shows fluorescence under a fluorescent microscope. In the indirect staining method, the antibody not labeled with fluorescein used in the first step plays a dual role, acting as an antibody to the antigen, and acting as an antigen against the anti-antibody of the second step. If an unknown antibody is examined, the antigen sample is the known serum to be tested as the first antibody, and the base step is the same as the test antigen.

Since immunoglobulins are species-specific, labeled anti-globulin antibodies must be prepared by immunizing other animals with the same animal serum globulin of the first antibody.

The advantage of the indirect staining method is that it can check both unknown antigens and known antibodies; with a labeled anti-globulin antibody, it can bind to antibodies of all animals of the same species and examine various unknown antigens or antibodies. High sensitivity. The disadvantage is that because there are many factors involved in the reaction, the possibility of interference is also large, and the judgment result is sometimes difficult, the operation is cumbersome, the control is more, and the time is long. The indirect method should be set up for yin and positive specimens, and an intermediate layer control should be provided (ie, the middle layer plus negative serum instead of positive serum).

3. Anti-complement staining

The anti-complement staining method, referred to as the complement method, is an improved method of indirect staining, first established by Goldwasser et al. This method utilizes the principle of complement binding reaction, and uses fluorescein to label anti-complement antibodies to identify unknown antigens or unknown antibodies (sera to be tested). The staining procedure is also divided into two steps: first unlabeled antibody and complement are added to the antigen sample, allowed to react, washed with water, and then labeled with anti-complement antibody. If the antigen antibody reacts in the first step to form a complex, the complement is bound by the antigen-antibody complex, and the fluorescein-labeled anti-complement antibody added in the second step specifically reacts with complement to form an antigen-antibody. - Complement-anti-complement antibody complex that fluoresces.

Anti-complement staining has the same advantages as indirect methods and, in addition, has the unique advantage of requiring only one labeled anti-complement antibody to detect various antigen-antibody systems. Because complement is not specific, it can react with any mammalian antigen-antibody system. Its disadvantage is that it has many components involved in the reaction, and the dyeing process is complicated and troublesome.

In addition to the above three methods, there are some methods that have evolved on the basis of this, such as a two-layer method, a sandwich method, a mixing method, a three-layer method, an antibody-anti-complement method, and the like.

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