Due to the complexity and diversity of ANA, there are many measurement methods. At present, the commonly used methods of ANA include immunofluorescence, radioimmunoassay, ELISA, immunodiffusion, convection immunoelectrophoresis and immunoblotting.
1. The most commonly used method for detecting total ANA in serum by immunofluorescence is fluorescence immunohistochemistry. Mice liver slices or slides are often used as the nuclear matrix. The results are relatively stable and reliable. The fluorescence staining of the nuclear nuclei seen under a fluorescent microscope is a positive reaction. If the patient's serum is first diluted in different proportions, a rough quantitative test can be done. When the 1:80 dilution is still positive, it has a greater reference value for the diagnosis of SLE. Observation results under oil microscope can divide the ANA-positive fluorescence phenomenon into 4 main fluorescence karyotypes. The determination of karyotype has further reference value for clinical diagnosis. â‘ Peripheral type indicates the presence of anti-DNA antibodies; â‘¡Homogeneous type indicates the presence of anti-DNP antibodies; â‘¢Spot (particle) type is mostly anti-ENA antibody; â‘£Nuclear type is mostly anti-nucleosome antibody. SLE patients often have peripheral type, homogeneous type or mixed type, spot type is more common in mixed connective tissue disease, and scleroderma is mostly nucleolar type; peripheral type has a high specificity for SLE.
In recent years, someone has used trypanosomes or blood flagellates (such as the green fly short film test) as a substrate to determine anti-dsDNA antibodies, because the blood matrix of these blood parasites contains a large amount of pure dsNDA, without other antigen interference; It can be seen that the moving matrix at the end of the flagella shows clear fluorescence. Therefore, this test has the advantages of strong specificity and high sensitivity for the determination of anti-dsDNA antibodies. In addition, the short membrane worm is harmless to humans and animals and can be cultured artificially. The source is convenient and worth promoting.
2. Radioimmunoassay is commonly used to detect anti-DNA antibodies, including Farr method and filtration method. ①The principle of Farr method is to label DNA with isotope, the labeled DNA is combined with the anti-DNA antibody of the tested serum, precipitated by 50% ammonium sulfate saturated solution, and then compare the radioactivity in the precipitate and the supernatant to obtain DNA The binding activity is generally positive if the binding rate is greater than 20%. ②The filtration method is to filter with a cellulose ester membrane filter (pore size 0.45μm) when separating the conjugates. The free DNA is filtered and the complex bound to the antibody is retained on the filter membrane.
3. Clinically, ELISA mainly uses indirect ELISA to detect anti-dsDNA antibodies, and its repeatability and sensitivity are higher than that of convection immunoelectrophoresis and double diffusion. Kits are currently available.
4. In immunoblotting, the mixed antigens are first subjected to gel electrophoresis to separate different zones, and then these bands are transferred to a nitrocellulose membrane, and finally labeled with acid-labeled antibodies or radioisotope antibodies for detection and analysis. . Since the test does not require a single antigen to be purified, it can be analyzed and tested on the same solid phase with high sensitivity and strong specificity. Therefore, it has been widely used in the detection of various autoantibodies in the serum of patients with autoimmune diseases, such as the detection of anti-Sm antibodies, anti-RNP antibodies, anti-SS-A antibodies, and SS-B antibodies.
In addition, traditional agar two-way diffusion method, convection immunoelectrophoresis method, indirect hemagglutination method and complement fixation test can also be used.
The experimental conditions required for these tests are relatively low, but the sensitivity is also relatively low.
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