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_sciences_  
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**Luminex Multiplex Bead Assay Monitoring HLA IgG**  
**Antibodies in Sensitized Pre- and Post-Transplant Patients:**  
**Clonality of the Detection Antibody Impacts Specificity**  
**and Sensitivity**  
**[PERSON] 1,2,*\({}^{\copyright}\), [PERSON] 3 and [PERSON] 4**  
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suggest possible pitfalls in the immunoassay, and raise concerns about the specificity and sensitivity of the assay for the reliable measurement of the levels of HLA Abs. HLA molecules on the cell surface occur as hetero-dimers for HLA-I (\(\alpha\)-chain and \(\beta\)2-microglobulin \(\beta\)2m) and HLA-II (\(\alpha\)- and \(\beta\)-chains), complexed with a short peptide. These trimeric structures are designated as \"HLA closed conformers (HLA CC)\" [23]. These heteromeric HLA are involved in antigen presentation. In addition to trimers, HLA molecules on the cell surface also occur as monomeric heavy chains (HC), called \"HLA open conformers (HLA OC)\" [24]. HLA OC are expressed on the surface of metabolically activated cells, including human T lymphocytes, EBV-transformed B cells, CD19+ B cells, CD8+ T cells, CD56+ NK cells, CD14+ monocytes, extravillous trophoblasts and monocytes, B-cell lines (RAJI, NALM6), and the myeloid cell line (KG-1A) [25; 26; 27; 28; 29; 30; 31]. These studies also show that the tyrosyl and seryl residues, in the extended cytoplasmic tail of naturally occurring HLA-I OC, can be phosphorylated and involved in signal transduction [32]. HLA Abs, associated with pre- and post-transplant sensitization [8; 9; 10; 12; 13], are monitored with Luminex-based multiplex single-antigen bead immunoassay (SAB immunoassay assay), in which SABs are coated with individual HLA molecules. A vast majority of reports have observed HLA Abs using single-antigen SABs (LABScreen, LS) from a vendor. Some of the studies aimed to distinguish Abs binding to HLA-I OC, induced by acid or heat treatment, from those binding to HLA CC (regular beads), which showed that Abs against intact HLA CC, but not those against denatured HLA OC, are predictive of graft failure [11; 16; 17; 18; 19; 20; 21; 22]. In addition to LS-SAB, another SAB (LIFECODES, LC) from a different vendor is used routinely outside the USA [33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43]. We have compared the structural variants of the HLA-I molecules on LS-SAB and LC-SAB using the following two monoclonal Abs: mAb W6/32 (HLA trimer/CC-specific) and mAb TFL-006 (HLA monomer/OC-specific) [44]. The LS-SABs were positive for both W6/32 and TFL-006 [44; 45; 46], which is indicative of the presence of HLA CC admixed with HLA OC. In contrast, the LC-SABs were positive for W6/32, but negative for TFL-006, confirming the absence of HLA OC on LC-SAB [44; 45; 46]. Similarly, HLA-II-coated LS-SABs also carry an admixture of OC with CC. Monomeric HLA-II OC was documented when the anti-HLA-II IgG reactivity was detected in sera (n = 141) using the LS-SAB coated with HLA-DRB1*09:01, DRB3*01:01, DRB3*02:02, DRB3*03:01, DPB1*02:01, DPB1*20:01, and DPB1*28:01 [47]. The sera reacting to the LS-SAB failed to react with the native cell surface HLA (e.g., HLA-DRB1*09:01) in a flow cytometry crossmatch (FCXM) and in absorption/elution experiments, suggesting that some HLA-II alleles on the LS-SAB may be present as OC [48]. Several reports have documented many DSA monitored with LS-SABs in pre- or post-transplant sera were not associated with graft failure [11; 16; 17; 18; 19; 20; 21; 22; 49; 50].