Detection of antinuclear antibodies in serum by immunofluorescence technique assay
Detection of antinuclear antibodies in serum by immunofluorescence technique assay
Antinuclear antibody (ANA) is an autoantibody directed against the nuclear components of cells, with no organ or germline specificity. A positive ANA is indicative of autoimmune diseases such as SLE or rheumatism. Immunofluorescence (immunofluorescence) labeling technology is based on the principle of immunological antigen-antibody specific reaction, the application of fluorescein labeling antigen or antibody, detection of the target antigen or antibody method, commonly used in tissue or cellular antigen or antibody detection, but also can be used for liquid-phase antigen or antibody detection, such as immunofluorescence antibody technology, flow cytometry and so on. Because immunofluorescence technology is specific, sensitive, fast and simple, it is widely used in clinical pathology diagnosis and testing.
Principle
The basic principle of the immunofluorescence technique for the detection of antinuclear antibodies in serum is to add drops of serum to be tested (> 1:100 dilution) to a biological thin section (Hep-2 cells + frozen section of primate liver), the ANA in the serum binds specifically to the antigenic components of the nucleus, and the unbound free antibody is removed after washing. The formed immune complexes suggest the presence of ANA in the serum by binding to FITC-labeled goat anti-human IgG, and the nuclei show yellow-green fluorescence under fluorescence microscopy.
Operation method
Detection of antinuclear antibodies in serum by immunofluorescence technique assay
Principle
The basic principle of the immunofluorescence technique for the detection of antinuclear antibodies in serum is to add drops of serum to be tested (>1:100 dilution) to a biological thin section (Hep-2 cells + frozen section of primate liver), the ANA in the serum binds specifically to the antigenic components of the nucleus, and the unbound free antibody is removed after washing. The formed immune complexes suggest the presence of ANA in the serum by binding to FITC-labeled goat anti-human IgG, and the nuclei show yellow-green fluorescence under fluorescence microscopy.
Materials and Instruments
Equipment: Move The basic procedure for the detection of antinuclear antibodies in serum by immunofluorescence technique can be divided into the following steps: Caveat 1 If the background is dark, wash with 0.05% Tween 20-PBS to remove non-specific binding.2 Protect from light after addition of fluorescein-labeled antibody to prevent fluorescence quenching.3 Set up positive and negative control groups. For more product details, please visit Aladdin Scientific website.
① sealing medium and coverslip, fluorescence microscope, serum to be examined
② Positive serum (serum of SLE patients), negative serum (serum of healthy people)
③ Bio-sheets (coated with Hep-2 cells and monkey liver respectively)
Reagents:
① 0.05% Tween20/PBS, pH 7.4 Wash solution
② FITC-goat anti-human IgG antibody
A. Take a biopancake and add 25 μl of 1:100 diluted patient serum for 30 minutes at room temperature.
B. Remove the biopancake, rinse it once with PBS, soak it in PBS for 5 minutes, and drain it on a filter paper.
C. Add 20 μl of FITC-goat anti-human IgG fluorescent antibody for 30 minutes at room temperature.
D. Remove and wash it as in (2). Remove the biological thin section and wash as in (2).
E. Seal the section with a drop of phosphate-buffered glycerol and observe under the microscope.