Antibodies produced in specific autoimmune diseases can be screened on an appropriate tissue using indirect immunofluorescence techniques.
PRINCIPLE: Autoantibodies, which attach to the appropriate antigen during an initial incubation with substrate tissue, can be detected by an immunoglobulin reagent (IgG, IgA or IgM), which has been conjugated with a fluorochrome for visualization under UV light following excitation.
ROUTINE SUBSTRATE SLIDES (commercially available)
Primate cerebellum can be used to detect the following antibodies:- GAD, PCA (Yo), ANNA1 (Hu), ANNA2 (Ri), Ma/Ta, amphiphysin, CV2/CRMP5, AGNA, and Tr.
- Diluted serum is incubated with tissue section in a moist chamber at room temperature.
- Then unbound antibodies are removed by washing the slides in PBS.
- Sections are incubated with diluted anti-human immunoglobulins (monkey adsorbed)-FITC at room temperature.
- The FITC conjugate is washed off as above.
- The sections are mounted under a glass cover slip using buffered glycerol +DABCO.
- The slides are ready for viewing under the fluorescence microscope.
- Pattern indicative of neuro-autoantibodies must be confirmed by other means (Western blot etc) and results disseminated to the appropriate physician as soon as possible.