Aim: We aimed to determine the reactivity of DR17/DR18 antibody to DRB1*03:05 and address antigen equivalency discrepancies.
Method: We performed single antigen bead (SAB) test for class II at neat and multiple dilutions. We performed surrogate flow cytometry crossmatch (FCXM) with an HLA-DRB1*03:05 donor and compared to results from donors with HLA typing of DRB1*03:01/02.
Results: Mature protein sequence alignment for DRB1*03:05, DRB1*03:01 (DR17) and DRB1*03:02 (DR18) shows that DRB1*03:05 differs from DRB1*03:01 only at position 86 where glycine is replaced by valine. In contrast, DRB1*03:02 has phenylalanine, glutamic acid, and tyrosine at positions 26, 28, and 47 (Figure 2). We screened 705 samples with DR17/DR18 reactivity on SAB II and found MFIs to be of comparable strengths amongst the two beads representing these single antigens in all cases. We identified a DR17/DR18-positive serum with strengths of 7,222 MFI and 5,087 MFI respectively and performed SAB II at 1:2, 1:4 and 1:8 to determine if they diluted out differently, which would indicate they are different specificities. We found reactivity remained similar between the DR17 and DR18 beads at all dilutions (Table 1), implicating little difference between the two antigens at the serological level. We further confirmed this result in a functional assay using surrogate donor cells with DRB1*03:05 typing and compared these results to reactivity seen with surrogate donor cells with DRB1*03:01 or DRB1*03:02 typing. The sera produced similar FCXM results with all three donor cells (Table 1), implicating little difference between the two antigens at the functional level.
DRB1 typing Neat 1:2 1:4
SAB II - DR17 (MFI) n/a 7,212 3,219 3,118
SAB II - DR18 (MFI) n/a 5,087 2,750 2,677
FCXM B-cell 03:05 Positive Negative Negative
FCXM B-cell 03:01 Positive Negative Negative
FCXM B-cell 03:02 Positive Negative Negative
Table 1: SAB and FCXM results
Conclusion: Our results indicate serological and functional reactivity is similar for DRB1*03:01, *03:02 and *03:05, and further suggests that DR17/18 are not different, and that the distinction between DR17/18 is unnecessary.
