Encyclopedia of Experiments: Immunology
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Transcript
Take a peptide array derived from an organ donor's human leukocyte antigen or HLA sequence comprising different peptides on cellulose membrane.
Each peptide carries at least one amino acid mismatch between the donor and recipient HLA sequence, representing a potential epitope for antibody-mediated organ rejection.
Treat with a blocking buffer to prevent non-specific antibody interaction.
Add recipient serum collected post-organ transplantation, containing alloantibodies.
These antibodies, formed against the HLA of the donor, bind to their target epitope.
Add a peroxidase-conjugated secondary antibody that binds to the alloantibody.
Add chemiluminescent substrate and hydrogen peroxide. The peroxidase utilizes hydrogen peroxide to oxidize the substrate, emitting light.
Scan the membrane to obtain an image.
Add a buffer to strip the bound antibodies. Reprobe with pre-transplantation recipient serum and obtain an image.
Compared to the pre-transplantation image, dark spots on the post-transplantation image indicate donor-specific HLA epitopes that incite alloantibody reactivity.
