Recombinant HLA-I (rHLA-I) molecules are the antigenic targets used to screen HLA-I DSA in the sera of pre- or post-transplant patients. A conformational diversity, β2-microglobulin-associated HLA-I heavy chain (β2aHC) and β2-microglobulin-free HLA-I heavy chain (β2fHC), of a single rHLA-I molecule may exist on the surface of Single Antigen Beads (SAB) assays. There is a need to assess the amount of β2fHC coated on SAB to help elucidating the nature of the DSA present in the sera of pre- or post-transplant patients.

Methods:

We used anti-HLA-I monoclonal antibodies (mAbs), targeting different conformations of the human MHC-I complex, W6/32 and TFL-006, specific for β2aHC and β2fHC respectively. We used a multiplex Luminex-based immunoassay to detect the mAbs IgG reactivity to HLA-I alleles. The SAB kits used were: HLA-classI Beads, Lot#8; and iBeads, Lot#1 (One Lambda). Both kits covered the same panel of rHLA-I antigens (31 HLA-A, 50 HLA-B, 16 HLA-Cw); however, HLA-classI Beads harbors both β2aHC and β2fHC rHLA-I molecules, whereas iBeads harbors β2aHC only. mAbs were tested at 10[micro]g/mL, MFI cutoff >1000.

Results:

The percentage of β2fHC, coated on SAB, is significantly different between HLA-A, -B and -Cw coated beads (p≤0.0001); HLA-Cw coated beads being highest (25±9%), followed by HLA-B (17±8%), and then HLA-A (11±7%). Therefore, rHLA-A heavy chains, associate more strongly with β2-microglobulin than rHLA-B and rHLA-Cw heavy chains. The table shows the reliable rHLA-I alleles for monitoring HLA-I DSA in both kits.

Conclusion:

The clinical monitoring of HLA-I DSA in the sera of pre- and post-transplant patients should be performed with caution when using HLA-classI Beads or iBeads, as some beads harbor both β2fHC and β2aHC and will detect DSA associated with negative Flow-XM. Therefore, the clinical evaluation of pre- or post-transplant DSA, using both kits, may improve the allocation of organ in patients awaiting a transplant and the therapeutic management of post-transplant patients, by discriminating β2fHC HLA-I DSA.

CITATION INFORMATION: Jucaud V, Ravindranath M, Terasaki P. Evaluation of Conformational Diversity of a Single HLA-I Molecule Coated on Single Antigen Beads: Potential Impact on Organ Allocation? Am J Transplant. 2016;16 (suppl 3).

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