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

In This Article

Abstract

Source: Liu, P., et al. Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation. J. Vis. Exp. (2017).

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 20x30=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 examples that we had tried, the 600 spot array can hold all the sequences generated from ~2 separate transplant cases.
1. Rationally plan the most eff....

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Results

Dot blot analysis, protein detection, experimental result, microarray visualization.
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 ill.......