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Custom Radioimmunoprecipitation Assay essay paper
Radioimmunoprecipitation assay is an in vitro technique to detect antigens or antibodies using antigen-antibody reaction principle. The technique was developed in the 1950’s by a Nobel Prize winner Rosalyn Yalow to study insulin concentrations (Yalow and Berson, 1960). Radioimmunoprecipitation assay is a highly sensitive method to reveal the interested antigen, however it has some considerable limitations that prevent its everyday application, such as the need to work with radioactive iodine and special equipment to provide safety. Thus, this test is mostly used in scientific laboratories. Moreover, anti-acetylcholine receptor antibodies (anti-AChR) may also be present in individuals with thymoma, and autoimmune liver disease (NHS Greater Glasgow and Clyde, 2013).
In myasthenia gravis, anti-AChR are present in high concentrations in the plasma. These antibodies are the pathophisiological basis for the disease development and progression of myasthenia as soon as they bind to the post-synaptic acetylcholine receptor preventing adequate synaptic signal transmitting (NHS Greater Glasgow and Clyde, 2013).
The method of radioimmunoprecipitation assay lies in 125I-labelled bungarotoxin (a neurotoxin that inhibits binding acetylcholine to corresponding receptors) incubation with test sera. Should anti-AChR be present in the patient, a antigen-antibody complex develops. The complex is precipitated with anti-human IgG and the centrifugated precipitate is tested for radioactivity. The steps of the test are the following (Buckley and Vincent, 2005; Matthews et al 2004):
a) add the serum with anti-AChR (marked Ag) to 125I-labelled bungarotoxin (marked Ab)
b) incubate at room temperature for 2 hours to bind anti-AChR to 125I-labelled bungarotoxin
c) add anti-human IgG to make the antigen-antibody complex insoluble and thus incubate at room temperature overnight
d) wash the precipitate, centrifuge and count for 125I in a gamma counter (not shown)
e) the concentration of anti-AChR is calculated according to the appropriate formular that accounts the test control.
In conclusion, the test requires one day to be completed and the anti-AChR concentration is given in nmol/L.