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

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.

Ramifications of the HLA-I Allelic Reactivity of Anti-HLA-E*01:01 and Anti-HLA-E*01:03 Heavy Chain Monoclonal Antibodies in Comparison with Anti-HLA-I IgG Reactivity in Non-Alloimmunized Males, Melanoma-Vaccine Recipients, and End-Stage Renal Disease Patients

“…The SAB products in every lot include 31 HLA-A, 50 HLA-B, and 16 HLA-C alleles. The beads supplied by the manufacturer have 2 categories of proteins attached to the beads [32,33,[37][38][39]: (1) HLA HC polypeptide only and (2) HC in association with β2m. The mean fluorescent intensity (MFI) values were obtained for IgG antibodies reacting to HLA-coated beads each allele of every HLA isomer.…”

“…Normalized MFI of mAbs were obtained as follows: ((Trimmed MFI-MFI of mAbs treated with PBS alone)-(negative control beads)). This is an essential step because when antibodies were tested on beads with PBS alone, differential MFI values were observed with different HLA molecules [37][38][39]. Each analysis included data obtained from >100 beads and was performed in duplicate.…”

Four Faces of Cell-Surface HLA Class-I: Their Antigenic and Immunogenic Divergence Generating Novel Targets for Vaccines

“…While investigating the HLA-E reactivity in alloimmunized normal males, polyreactive HLA-Ia reactivity was observed in anti-HLA-E-positive sera. These HLA-I polyreactive antibodies that are also formed during end stage organ diseases are particularly important from the perspective of HLA sensitization that occurs in patients with such diseases [70,71]. HLA allo-antibodies formed against allograft specific alleles that are present in the donor and not in the recipient can cause damage to the transplanted allograft.…”

“…However, the polyreactive HLA-I IgGs capable of reacting only with Face-2 of several HLA isoforms and their alleles will not react with Face-1 expressed on allograft endothelial cells. Therefore, the polyreactive IgGs may not be the ones involved in antibody-mediated destruction of allografts [70,71].…”

“…Adam K. Idica, a dynamic researcher from Terasaki Foundation Laboratory has developed a category of beads that contain only Face-1 but not Face-2, based on the trypsin assay of Demaria et al [61] for eliminating Face-2 but not Face-1 from the cell surface. Utilizing a modified version of the assay, One Lambda Inc. has generated beads carrying only Face-1, called iBeads, which are different from regular LABScreen SABs, in which Face-1 is admixed with Face-2 [56,71]. The LABScreen beads were commercialized for monitoring HLA antibodies in pre-and post-transplant patients' sera.…”