Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Apr;101(4):764-777.
doi: 10.1097/TP.0000000000001420.

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

Vadim Jucaud  1 , Mepur H Ravindranath, Paul I Terasaki
Affiliations

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

Vadim Jucaud et al. Transplantation. 2017 Apr.

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 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.

PubMed Disclaimer

Conflict of interest statement

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.

Figures

FIGURE 1.
FIGURE 1.
HLA-I beads. Anti–HLA-I mAbs (W6/32, TFL-006, HC-10) reactivity against all HLA-A (n = 31), HLA-B (n = 50) and HLA-Cw (n = 16) antigens in HLA-I beads. The MFI cutoff used for a positive reaction is 1000. Note that W6/32 binds to 100% of HLA-A, HLA-B, and HLA-Cw antigens and HC-10 binds to 39% of HLA-A, 94% of HLA-B and 100% of HLA-Cw antigens with high MFI. In contrast, TFL-006, though binds to 100% of HLA-A, HLA-B and HLA-Cw antigens, the MFI for the antigens remained lower than that of W6/32 and HC-10.
FIGURE 2.
FIGURE 2.
iBeads. Anti–HLA-I mAbs (W6/32, TFL-006, HC-10) reactivity against all HLA-A (n = 31), HLA-B (n = 50) and HLA-Cw (n = 16) antigens in iBeads. The MFI cutoff used for a positive reaction is 1000. W6/32 binds to 100% of HLA-A, HLA-B and HLA-Cw antigens with high MFI. TFL-006 poorly recognizes of HLA-A antigens (10%), while failing to recognize HLA-B and -Cw antigens (0%). HC-10 binds to 35% of HLA-A, 56% of HLA-B and 63% of HLA-Cw antigens.
FIGURE 3.
FIGURE 3.
Acid-treated beads. Anti–HLA-I mAbs (W6/32, TFL-006, HC-10) reactivity against all HLA-A (n = 31), HLA-B (n = 50) and HLA-Cw (n = 16) antigens in acid-treated beads. The MFI cutoff used for a positive reaction is 1000. W6/32 failed to recognize HLA-A, HLA-B and HLA-Cw in acid-treated beads (0%). TFL-006 shows 100% reactivity to the HLA-A, HLA-B and HLA-Cw antigens. HC-10 binds to 65% of HLA-A and 100% of HLA-B and HLA-Cw antigens.
FIGURE 4.
FIGURE 4.
Alkali-treated beads. Anti–HLA-I mAbs (W6/32, TFL-006, HC-10) reactivity against all HLA-A (n = 31), HLA-B (n = 50) and HLA-Cw (n = 16) antigens in alkali-treated beads. The MFI cutoff used for a positive reaction is 1000. The binding pattern of W6/32 and TFL-006 on alkali-treated beads are similar to the binding on acid treated beads. HC-10 binds to 45% of HLA-A on alkali treated beads, 94% of HLA-B and 100% of HLA-Cw on alkali-treated beads.
FIGURE 5.
FIGURE 5.
HLA-I beads lot-to-lot variability expressed as the difference between anti–HLA-I mAbs (W6/32, TFL-006, HC-10) reactivity against HLA-I beads lot 8 and lot 9 (lot 8 MFI – lot 9 MFI). Positive values illustrate higher reactivity with lot 8, while negative values illustrate higher reactivity with lot 9.
FIGURE 6.
FIGURE 6.
Density (%) of β2fHC coated on HLA-I beads and iBead, as assessed by the following formula: %β2fHC = (TFL-006 MFI)/(TFL-006 MFI + W6/32 MFI). Boxed antigens represent beads coated with non-detectable β2fHC with MFI < 1000 on HLA-I beads, whereas dotted-boxed antigens represent beads coated with detectable β2fHC with MFI > 1000 on iBeads.
FIGURE 7.
FIGURE 7.
Density (%) of pepF-β2aHC coated on iBeads as assessed by the following formula: %pepF-β2aHC = (HC-10 MFI)/(W6/32 MFI). Boxed antigens represent beads coated with nondetectable pepF-β2aHC with MFI < 1000 on iBeads.
FIGURE 8.
FIGURE 8.
A, Density of HLA-I conformational variants on SAB and T-cells. HLA-I beads has highest density of pepA-β2aHC followed by pepF-β2aHC and then low density of β2fHC. iBeads has highest density of pepA-β2aHC and low density pepF-β2aHC and minimal density of β2fHC. Acid/Alkali-treated beads only bear β2fHC. T-cells only bear pepA-β2aHC. B, HLA-I conformational variants recognized by the mAbs. W6/32 recognize both pepA-β2aHC and pepF-β2aHC. HC-10 recognize pepF-β2aHC and pepF-β2fHC. TFL-006 recognize pepF-β2fHC and possibly pepA-β2fHC.
See this image and copyright information in PMC

Comment in

  • Conformational Variants of HLA-I Antigens on Luminex Single Antigen Beads.
    Visentin J, Guidicelli G, Taupin JL. Visentin J, et al. Transplantation. 2017 Apr;101(4):e152-e153. doi: 10.1097/TP.0000000000001607. Transplantation. 2017. PMID: 27941435 Free PMC article. No abstract available.
  • The Authors' Reply.
    Ravindranath MH, Jucaud V. Ravindranath MH, et al. Transplantation. 2017 Apr;101(4):e153-e154. doi: 10.1097/TP.0000000000001629. Transplantation. 2017. PMID: 28323776 Free PMC article. No abstract available.

References

    1. Terasaki PI. Humoral theory of transplantation Am J Transplant 2003. 3665–673 - PubMed
    1. Pei R, Lee JH, Shih NJ. Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody specificities Transplantation 2003. 7543–49 - PubMed
    1. Bray RA, Nolen JD, Larsen C. Transplanting the highly sensitized patient: the emory algorithm Am J Transplant 2006. 62307–2315 - PubMed
    1. Bielmann D, Hönger G, Lutz D. Pretransplant risk assessment in renal allograft recipients using virtual crossmatching Am J Transplant 2007. 7626–632 - PubMed
    1. Cecka JM, Kucheryavaya AY, Reinsmoen NL. Calculated PRA: initial results show benefits for sensitized patients and a reduction in positive crossmatches Am J Transplant 2011. 11719–724 - PubMed

Publication types