Monitoring native HLA-I trimer specific antibodies in Luminex multiplex single antigen bead assay: Evaluation of beadsets from different manufacturers.

Luminex single antigen bead (SAB) assay utilizes beadsets coated with a set of cloned and purified HLA molecules, for monitoring serum anti-HLA antibodies. Particularly, the level of serum IgG against native HLA-I trimers (heavy chain (HC) and β2-microglobulin (β2m) with a peptide), expressed in allograft tissues is correlated with graft failure. In addition to native trimeric HLAI, the beadsets may carry HC only or the dimeric variants, peptide-free HC with β2m and β2m-free HC with or without peptides. Currently, three different HLA-I coated beadsets have been produced commercially. The HLA antigen density on one beadset was reported to be approximately 50% of that present on another beadset as evidenced by the binding of an anti-HLA-I mAb W6/32. To date, no efforts have been made to compare the relative distribution of HLA-I variants in these three beadsets. In this study, using monoclonal antibodies (W6/32, HC-10 and TFL-006) that can distinguish the structural variants based on their epitope specificities, the nature of the variants in the three beadsets were comparatively evaluated. One beadset (Beadset A, see Materials and methods for Brand and Manufacturer's names) (W6/32+/HC-10+/TFL-006+) carried at least three variants, while beadset B (W6/32+/HC-10+/TFL-006-) carried two (peptide-associated and peptide-free β2m-HC) and the beadset C (W6/32+/HC-10-/TFL-006-) carried exclusively the HLA-I trimer suggesting its usefulness for specific monitoring native HLA-I trimer antibodies. Because of the salient differences in the variants coated on the different beadsets, it would be warranted to investigate, if these differences are clinically relevant for monitoring serum anti-HLA antibodies in sensitized patients waiting for donor organs and in allograft recipients (274).