Luminex Multiplex Bead Assay Monitoring HLA IgG Antibodies in Sensitized Pre- and Post-transplant Patients: Clonality of the Detection Antibody Impacts Specificity and Sensitivity

Mepur H Ravindranath,Carly J Amato-Menker,Narendranath M Ravindranath

doi:10.3390/app11146430

Abstract

The number and the binding affinity, measured as the mean fluorescent intensity (MFI) of HLA-specific IgG antibodies, formed in the sera of end-stage organ disease patients and allograft recipients, referred to as sensitization, may restrict the availability of a donor organ and/or lead to graft failure after transplantation. The MFI of HLA Abs in sera is monitored with the Luminex-based single-antigen bead (SAB) immunoassay. The following two factors may impact the reliable measurement of MFI: one, the HLA structural variants on the SAB, namely, trimeric HLA (closed conformers, CC) and monomeric heavy chains (open conformers, OC); and two, the nature of the detection Abs, namely, IgG heavy-chain binding polyclonal-Fab (IgHPolyFab) or Fc-binding monoclonal-IgG (FcMonoIgG). Anti-CC Abs correlate with positive flow cross-matches, and are considered to be pathogenic and damaging to the graft, whereas anti-OC Abs appear to have little relevance to graft attrition. The presence of both CC and OC on beads may impair the reliability of monitoring the nature and MFI of pathogenic Abs. Our objective is to compare the MFI of the HLA Abs in the sera of 20 sensitized patients in two different SAB assays, with the two detection Abs. Our data reveal that the admixture of OC with CC on beads will affect the reliability of the measurement of the pathogenic Abs, and that FcMonoIgG is the more sensitive and specific detection Ab for the accurate assessment of HLA sensitization.

Highlights

The presence of pre-existing anti-HLA antibodies (Abs) is referred to as sensitization.It is one of the significant immunological barriers to organ transplantation
The level of sensitization in the sera of waitlist patients is assessed by the number of HLA antigens that are recognized by the serum IgG Abs, and by the mean fluorescent intensity (MFI) of anti-HLA Abs on LS-single-antigen bead (SAB) and LC-SAB, using two different detection Abs
The MFI of both HLA-I and HLA-II Abs differed between LS-SAB, LC-SABS, and the detection Ab

Summary

Introduction

The presence of pre-existing anti-HLA antibodies (Abs) is referred to as sensitization. It is one of the significant immunological barriers to organ transplantation. The level of sensitization is assessed based on the number of HLA antigens recognized by the serum IgG Abs, and on the MFI of the anti-HLA Abs at a particular dilution These Abs may (a) restrict the availability of a given organ for a particular patient, and are a significant cause of long-term graft attrition post-transplantation [8,9,10,11,12,13,14,15]; (b) react with mismatched intact HLA antigens on a donor organ (donor-specific Abs, DSA); and (c) recognize non-donor-specific alloHLA (NDSA).

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Conclusion