Development and Validation of a Multiplex Non-HLA Antibody Assay for the Screening of Kidney Transplant Recipients

Abstract

The best treatment for patients with end-stage renal disease is kidney transplantation. Although graft survival rates have improved in the last decades, patients still may lose their grafts partly due to the detrimental effects of donor-specific antibodies (DSA) against human leukocyte antigens (HLA) and to a lesser extent also by antibodies directed against non-HLA antigens expressed on the donor endothelium. Assays to detect anti-HLA antibodies are already in use for many years and have been proven useful for transplant risk stratification. Currently, there is a need for assays to additionally detect multiple non-HLA antibodies simultaneously in order to study their clinical relevance in solid organ transplantation. This study describes the development, technical details and validation of a high-throughput multiplex assay for the detection of antibodies against 14 non-HLA antigens coupled directly to MagPlex microspheres or indirectly via a HaloTag. The non-HLA antigens have been selected based on a literature search in patients with kidney disease or following transplantation. Due to the flexibility of the assay, this approach can be used to include alternative antigens and can also be used for screening of other organ transplant recipients, such as heart and lung.

Antibodies against ARHGDIB are associated with long-term kidney graft loss

“…This strategy was chosen because the coupling process may influence the conformation and accessibility of epitopes predominantly recognized on non-HLA beads. 11 Previously we found that the correlation between results obtained with our test and with commercial assays depended on the coupling method of the proteins. This demonstrates the necessity of complete transparency and detailed description of methods when comparing the effects of non-HLA antibodies in patient groups.…”

“…11 Antibodies against the glomerular basement membrane protein agrin have been described in the context of transplant glomerulopathy. 11 Antibodies against the glomerular basement membrane protein agrin have been described in the context of transplant glomerulopathy.…”

“…We selected 14 non-HLA target proteins from the literature. 11 Antibodies against the glomerular basement membrane protein agrin have been described in the context of transplant glomerulopathy. 12 Antibodies against adipocyte plasma membrane-associated protein (APMAP), Rho GDP-dissociation inhibitor 2 (ARHGDIB), Rho guanine nucleotide exchange factor 6 (ARHGEF6), Lamin B1, BPI fold-containing family B member 1 (LPLUNC1), protein kinase C zeta type (PRKCZ), and tubulin beta-4B (Tubb4B) were all demonstrated either in chronic hemodialysis patients or patients awaiting kidney transplantation.…”

“…In this retrospective study on a large national cohort of 4770 renal transplant recipients, we assessed the impact on graft survival of IgG autoantibodies against 14 previously identified target proteins. 11…”

“…This strategy was chosen because the coupling process may influence the conformation and accessibility of epitopes predominantly recognized on non-HLA beads 11. After binding, anti-ARHGDIB antibodies may initiate complement activation causing local inflammation, which may explain why a relation between their presence and graft loss was observed only after deceased-donor transplantation.In our study, non-HLA proteins were coated directly or indirectly (via HaloTag) to the microspheres.…”