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

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.