A Peptide Array-Based Detection of Anti-donor HLA Alloantibodies in Organ Recipients

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.

When the arrays have been synthesized, block the membranes with 20 milliliters of 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween 20 or TBST for the appropriate incubation period with rocking. Next, wash the membrane three times with 20 milliliters of fresh TBST for 5 minutes per wash, followed by incubation with 20 microliters of crude recipient serum in 2.5% milk in TBST for 2 to 3 hours at room temperature.

At the end of the incubation, wash the membrane three times for 10 minutes per wash and incubate the membranes with goat anti-human horseradish peroxidase-conjugated IgG secondary antibody for two hours.

Remove the unbound secondary antibody with three 10-minute washes in TBST, and develop the membranes in 5 milliliters of freshly prepared luminol solution plus 5 milliliters of peroxide solution. After one minute, visualize the enhanced chemiluminescence signals on a suitable imager.

After saving the developed images, incubate the membranes with 20 milliliters of commercial stripping buffer at 37 degrees Celsius for 20 minutes followed by three 10-minute washes in TBST. Then block, reprobe, and visualize the membranes as just demonstrated with a serum sample from the same patient obtained at a different time point.