This video demonstrates an assay to detect anti-donor HLA alloantibodies in organ recipients. A peptide array is synthesized to represent potential epitopes for antibody-mediated organ rejection. The array is incubated with the recipient's serum, containing alloantibodies that bind to their target epitopes. Antibody binding is detected using chemiluminescence to identify donor-specific HLA epitopes that incite alloantibody reactivity.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Design of Custom Array Layout and Production Generate a spreadsheet of peptide sequences with corresponding allele callings. The array has 20 rows and 30 columns and can hold up to 20×30=600 spots for peptides. Depending on the total number of peptides recorded in step 1.5 from one particular donor, and based on our past experience with two examp…
Representative Results
Figure 1. Image of an array section stained with Ponceau S Note disparity in color intensity is due to differences in amino acid composition among peptides, while peptide concentrations among all spot areas are the same. (The image is an illustrative example, not the one from this actual study.) <img alt="Fig…