Dataset Information

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

ABSTRACT: 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 ?2-microglobulin-associated HLA HC (pepA-?2aHC), peptide-free-?2aHC (pepF-?2aHC), and ?2-free HC (?2fHC) on every single antigen-coated bead. In contrast, iBeads harbor a high density of pepA-?2aHC, low density of pepF-?2aHC, and are lacking ?2fHC. The FCXM analyses confirmed the prevalence of pepA-?2aHC, but not pepF-?2aHC or ?2fHC 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 ?2fHC or pepF-?2aHC normalized donor-specific antibody level would reveal the true anti-pepA-?2aHC reactivity associated with positive FCXM.

SUBMITTER: Jucaud V

PROVIDER: S-EPMC7228605 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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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 V Ravindranath Mepur H MH Terasaki Paul I PI

Transplantation 20170401 4

<h4>Background</h4>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 patient ...[more]

PMID: 27495776

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Project description:IntroductionIn solid-organ transplantation, human leukocyte antigen (HLA) donor-specific antibodies (DSA) are strongly associated with graft rejection, graft loss, and patient death. The predominant tests used for detecting HLA DSA before and after solid-organ transplantation are HLA single antigen bead (SAB) assays. However, SAB assays may not detect antibodies directed against HLA epitopes that are not represented in the SAB. The prevalence and potential impact of unrepresented HLA epitopes are expected to vary by ethnicity, but have not been thoroughly investigated. To address this knowledge gap, HLA allele frequencies from seven ethnic populations were compared with HLA proteins present in SAB products from two manufacturers to determine unrepresented HLA proteins.MaterialsAllele frequencies were obtained from the Common, Intermediate, and Well Documented HLA catalog v3.0, and frequencies of unrepresented HLA types were calculated. Next-generation sequencing was used to determine HLA types of 60 deceased solid-organ donors, and results were used to determine if their HLA-A, -B, -C, and -DRB1 proteins were not present in SAB reagents from two vendors. Unrepresented HLA proteins were compared with the most similar protein in SAB assays from either vendor and then visualized using modeling software to assess potential HLA epitopes.ResultsFor the seven ethnic populations, 0.5% to 11.8% of each population had HLA proteins not included in SAB assays from one vendor. Non-European populations had greater numbers of unrepresented alleles. Among the deceased donors, 26.7% (16/60) had at least one unrepresented HLA-A, -B, -C, or -DRB1 protein. Structural modeling demonstrated that a subset of these had potential HLA epitopes that are solvent accessible amino acid mismatches and are likely to be accessible to B cell receptors.DiscussionIn conclusion, SAB assays cannot completely rule out the presence of HLA DSA. HLA epitopes not represented in those assays vary by ethnicity and should not be overlooked, especially in non-European populations. Allele-level HLA typing can help determine the potential for HLA antibodies that could evade detection.

Project description:Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i) using secondary antibodies designed to bind only non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs from the homogenates, and iv) using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes several solutions to avoid non-specific results. We strongly recommend the use of negative (i.e., TKO) and positive (i.e., hypothermic) controls in all experiments.

Dataset Information

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

Publications

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

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