High-throughput allogeneic antibody detection using protein microarrays

Abstract

Enzyme-linked immunosorbent assays (ELISAs) have traditionally been used to detect alloantibodies in patient plasma samples post hematopoietic cell transplantation (HCT); however, protein microarrays have the potential to be multiplexed, more sensitive, and higher throughput than ELISAs. Here, we describe the development of a novel and sensitive microarray method for detection of allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome, called HY antigens. Six microarray surfaces were tested for their ability to bind recombinant protein and peptide HY antigens. Significant allogeneic immune responses were determined in male patients with female donors by considering normal male donor responses as baseline. HY microarray results were also compared with our previous ELISA results. Our overall goal was to maximize antibody detection for both recombinant protein and peptide epitopes. For detection of HY antigens, the Epoxy (Schott) protein microarray surface was both most sensitive and reliable and has become the standard surface in our microarray platform.

A confirmation of chronic graft-versus-host disease prediction using allogeneic HY antibodies following sex-mismatched hematopoietic cell transplantation

“…Antibody detection and threshold calculation was performed according to our previously published protein microarray platform. 5 Full details of methods and statistical analyses can be found in the Online Supplementary Appendix. There was no appreciable difference in seropositivity cutoffs and antibody quantifications between plasma and sera samples (Online Supplementary Figure S1).…”

“…Jed Paul, 1 Hideki Nakasone, 2 Bita Sahaf, 1 Fang Wu, 1 Kathy Wang, 3 Vincent Ho, 3 Juan Wu, 4 Haesook Kim, 3 Bruce Blazar, 5 Jerome Ritz, 3 Alan Howard, 6 Corey Cutler, 3 and David Miklos…”