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

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 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…

Materials

Peptide array	INTAVIS Bioanalytical Instruments
Ethanol	Sigma-Aldrich	E7023
Ponceau S solution	Sigma-Aldrich	P7170
Non-fat milk	Bio Rad Laboratories	1706404
TBST	Santa Cruz Biotechnology	10711454001
Goat anti-human IgG–HRP	ThermoFisher Scientific	A18811
Clarity Western ECL Substrate	Bio Rad Laboratories	1705061
Restore Western Blot Stripping Buffer	Thermo Scientifics	21059
ChemiDoc gel imaging system	Bio Rad Laboratories	1708265