Encyclopedia of Experiments: Immunology
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Transcript
To detect islet autoantibodies — a biomarker of type-1 diabetes — take human serum samples and add acetic acid to inhibit interfering components.
Incubate and then add an alkaline buffer to neutralize the acid.
Transfer the treated serum to a multi-well plate containing a mixture of human islet autoantigens, including biotinylated capture and ruthenium-conjugated antigens, each at an optimal concentration for the assay.
The islet autoantibody in the positive sample bridges the ruthenium-conjugated antigen to the biotinylated capture antigen, forming an antigen-antibody complex.
Transfer the mixture into a streptavidin-coated electrochemiluminescence plate pre-treated with a blocking agent and equipped with working and counter electrodes.
Incubate to allow biotinylated complex interactions with the bound streptavidin and then wash.
Add a buffer containing tripropylamine or TPA.
In an electrochemiluminescence instrument, the voltage application between the electrodes initiates the oxidation of ruthenium and TPA, generating TPA radicals.
The TPA radicals excite ruthenium complexes, which emit photons while returning to their ground state.
A high chemiluminescence in the positive sample indicates the presence of islet autoantibodies.
