ravo Stiff-Person Blot
Recombinant immunoblot for the detection of GAD65- and Amphiphysin-autoantibodies for the diagnosis ofStiff-Person-Syndrome
Stiff-Person Syndrome is a rare neurological autoimmune disease that is character-ized by rigidity and episodic spasms of muscles as a result of continuous motor unit activity.
Antibodies to GAD (glutamat-decarboxylase) are considered as serological markers for Stiff-Person-Syndrome. The majority of patients have high titers of antibodies to both isoforms, GAD65 and GAD67. These enzymes catalyse the conversation of glutamate to GABA (ã-aminobutyric acid), a major inhibitory neurotransmitter of the CNS.
Amphiphysin expression is found in synaptic vesicels of neurons as well as in the sceletal musculature. The presence of anti-Amphiphysin antibodies (with or without GAD) indicates a paraneoplastic neurological syndrome caused by an underlying tumour.
Procedure (Short Description):
• Cover one strip with the ready to use positive and negative control (2 ml).
• Cover the other strips with 1.5 ml ready to use dilution buffer.
• Add 15 µl of specimen and mix carefully (end-dilution: 1 : 100).
• Incubate for 60 minutes at room temperature on a rocking table.
• Wash five times with diluted wash buffer.
strip. Specimen and negative control: add 2 ml alkaline phosphatase IgG
conjugate, ready to use, per strip.
• Incubate for 30 minutes at room temperature on a rocking table.
• Wash five times with diluted wash buffer.
• Cover each strip with 2 ml ready to use substrate-solution.
• Incubate for 25 minutes at room temperature until the bands become clearly
visible. See control scan for comparison.
• Transfer the strips to distilled water to stop the reaction. Put the strips onto
filter paper and let them dry. Store the strips in the dark.
back