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Conformational Variants of the Individual HLA-I Antigens on Luminex Single Antigen Beads Used in Monitoring HLA Antibodies: Problems and Solutions

Jucaud, Vadim BSc; Ravindranath, Mepur H. MSc, PhD; Terasaki, Paul I. PhD

doi: 10.1097/TP.0000000000001420
Original Basic Science—General

Background Single antigen beads (SAB) are used for monitoring HLA antibodies in pretransplant and posttransplant patients despite the discrepancy between virtual and actual crossmatch results and transplant outcomes. This discrepancy can be attributed to the presence of conformational variants of HLA-I on SAB, assessment of which would increase the concordance between SAB and flow cytometry crossmatch (FCXM) results, thus enabling improved organ accessibility for the waiting list patients and a better prediction of antibody-mediated rejection.

Methods The conformational variants were examined on HLA-I beads, iBeads, acid-/alkali-treated beads, and T cells using HLA-I monoclonal antibodies (W6/32, TFL-006, and heavy chain (HC)-10).

Results The affinity of the monoclonal antibodies against HLA-I beads confirmed the presence and heterogeneous density of peptide-associated β2-microglobulin–associated HLA HC (pepA-β2aHC), peptide-free-β2aHC (pepF-β2aHC), and β2-free HC (β2fHC) on every single antigen-coated bead. In contrast, iBeads harbor a high density of pepA-β2aHC, low density of pepF-β2aHC, and are lacking β2fHC. The FCXM analyses confirmed the prevalence of pepA-β2aHC, but not pepF-β2aHC or β2fHC on resting T cells.

Conclusions The strength of a donor-specific antibody should be assessed with a bead-specific mean fluorescence intensity cutoff based on TFL-006 reactivity against HLA-I beads, and HC-10 against iBeads, where the β2fHC or pepF-β2aHC normalized donor-specific antibody level would reveal the true anti–pepA-β2aHC reactivity associated with positive FCXM.

In order to distinguish between relevant and irrelevant HLA antibodies, the authors study the role of antibodies to conformational variants of HLA-I and show that the presence of these antibodies should be tested in order to assess correctly the relevant strength a given antibody.

1 Terasaki Foundation Laboratory, Los Angeles, CA.

Received 4 May 2016. Revision received 14 June 2016.

Accepted 4 July 2016.

Professor Paul Ichiro Terasaki passed away on January 25, 2016.

The entire project is funded by Terasaki Family Foundation.

V.J. has no conflicts of interest to disclose. M.H..R. and the late P.I.T., as coinventors, have filed US and European patent applications on HLA-I polyreactive anti–HLA-E mAbs, which include mAb TFL-006.

P.I. initiated this project, discussed its plan and offered advice during the early phases of this investigation. V.J. designed and carried out all the experiments using 3 different monoclonal antibodies and 4 different single-antigen beads, analyzed the data in comparison with previous publications, prepared the tables and figures, wrote the first draft of the article meticulously. M.H.R., based on discussion with the third author, initiated the investigation on the use the 3 monoclonal antibodies on Luminex single antigen beads and on T cells to elucidate variations within a single HLA allele, examined the data, discussed periodically with the first author and contributed to the writing and editing of the article. The late P.I.T. initiated a discussion with the second author about the importance of defining the variations of individual HLA antigens on the Bead using the monoclonal antibody developed at Terasaki Foundation Laboratory (TFL-006) along with mAbs W6/32 and heavy chain (HC)-10. During early phases of data collection, he discussed with the second author highlighting the importance of iBeads for monitoring sera of pre and posttransplant patients' sera.

Correspondence: Mepur H. Ravindranath, MSc, PhD, Terasaki Foundation Laboratory, Los Angeles, CA 90064. (

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