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Aim To evaluate variations of the expression of antibody epitopes associated with DQB1*03:01 alleles in B cells. Methods Antibody tests were performed using Luminex Single Antigen Bead (SAB) assay on serum samples treated with DTT. XMs were performed by flow cytometry, using lymphocytes from peripheral blood of healthy volunteers, which were treated with pronase. Results Four sera reacted strongly with beads carrying the following alleles: DQB1*03:01-DQA1*06:01, DQB1*03:01-DQA1*05:05, and DQB1*03:01-DQA1*05:03 (Figure 1). Sera JM and RH reacted strongly with B cells from all 6 donors who were positive for DQB1*03:01 indicating that these cells express adequate amount of DQ molecules (Figure 2A). However, XMs of sera CG and SR with the 4 heterozygous DQB1*03:01 B cells showed weak or no reactivity respectively (Figure 2B). In comparison to XM reactivity from sera JM and RH the XMs with sera SR and CG exhibited a 25-50-fold lower Δ MESF values (Figure 2A versus 2B). This unexpected low reactivity of sera SR and CG with B cells is striking since their reactivity with SAB is similar to that of serum JM except with the DQB1*03:01-DQA1*03:01 allele (Figure 1). Notably, SR and CG reacted with B cells that were homozygous for DQB1*03:01 (Figure 2B, Donor 1 and 2) indicating that the reactivity of these sera with the SAB was not due to denatured epitopes. Conclusions These results suggest that the observed variable expression of DQB1*03:01 epitope on B cells could be due to variation in the conformation of this epitope on the same DQ molecules with a potential impact on antibody affinity to this epitope. The discrepancy between SAB MFI values and XM reactivity is important to consider in assessing HLA compatibility and lymphocyte crossmatching.