1. immunofluorescence technique
Immunofluorescence technique is a method to detect the corresponding antigen (or antibody) in tissues, cells or serum by using fluorescein labeled antibody (or antigen). Fluorescent antibody is widely used in immunofluorescence detection and flow cytometry because of its safety and sensitivity. According to different labeling methods of fluorescein, it can be divided into direct fluorescent antibody and indirect fluorescent antibody. The first antibody among the inter-labeled fluorescent antibodies is not directly connected with fluorescein, but first combines the first antibody with protein, and then combines the second antibody with fluorescein with the first antibody. Through the combination of the second antibody, the signal can be amplified, so the sensitivity of detection can be improved to a certain extent, but the high background also reduces the specificity of detection. In recent years, with the continuous progress of fluorescein and fluorescence detection technology, the sensitivity of fluorescence detection has approached the level of isotope detection, and directly labeled fluorescent antibodies have gradually replaced indirectly labeled antibodies. These fluorescein-labeled antibodies directly bind to antigens, which greatly improves the specificity of detection and makes the detection results more accurate and reliable. With the development of fluorescence detection technology, immunofluorescence technology has been widely used in the diagnosis of infectious diseases, such as checking IgM antibodies in bacteria, viruses and spirochetes, as a sign of recent contact with antigens. Identification of lymphocyte subtypes by monoclonal fluorescence directly labeled antibody. By flow cytometry, different fluorescent antibodies can be used to stain the same cell with different antigens on the cell surface.
2. Radioimmunoassay
Radioimmunoassay is the most sensitive detection technology at present. Labeling antigen (or antibody) with radioactive isotope, and then combining with corresponding antibody (or antigen) to determine the radioactive detection result of antigen-antibody conjugate. Radioisotopes have pg-level sensitivity, and trace substances can be quantitatively detected by repeated irradiation. However, the harm of radioactive isotopes to human body also limits the use of this method.
3. Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay is the most widely used immunoassay method at present. This method combines the specificity of enzyme, antigen-antibody reaction and the effect of enzyme catalyzing substrate, and judges the detection result according to the color change after the enzyme acts on the substrate, and its sensitivity can reach ng level. Commonly used enzymes for labeling are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Enzyme-linked immunosorbent assay (ELISA) is widely used in disease detection because it does not need special instruments and is simple to detect. Commonly used methods include indirect method, sandwich method and BAS -ELISA. The indirect method is to put the protein to be detected in an orifice plate, then add the primary antibody, the enzyme-labeled secondary antibody and the substrate in turn, and quantitatively detect the antigen with an instrument (such as an enzyme-labeled instrument). This method is simple to operate, but its specificity is poor because of its high background. It has been gradually replaced by sandwich method. Sandwich method uses two kinds of primary antibodies to capture and immobilize the target antigen, which greatly improves the specificity of the reaction while ensuring the sensitivity. In recent years, the quantitative detection technology of antigen is also constantly innovating. In recent years, a multi-antigen detection kit has been developed based on sandwich ELISA, which can simultaneously detect the contents of multiple antigens in micro-liquid samples. The application of this technology greatly shortens the diagnosis time and improves the reliability and timeliness of diagnosis.
4. Immunocolloidal gold technology
After more than 30 years of development, colloidal gold technology is becoming more and more mature. In this method, colloidal gold particles are used to label the secondary antibody, and finally the secondary antibody labeled with colloidal gold is adsorbed on the filter membrane by using the specific reaction between antigen and antibody. This method is simple and rapid, and can be widely used in clinical screening.