Background: The blood group antigens S and s are defined by amino acids Met or Thr at position 29, respectively, on glycophorin B (GPB). Commercial anti-s reagents are expensive to produce because of the scarcity of human anti-s serum. Our aim was to develop hybridoma cell lines that secrete reagent-grade anti-s monoclonal antibodies (MoAbs) to supplement the supply of human anti-s reagents.
Study design and methods: Mice were immunized with the GPB(s) peptide sequence TKSTISSQTNGETGQLVHRF. Hybridomas were produced by fusing mouse splenocytes with mouse myeloma cells (X63.Ag8.653). Screening for antibody production was done on microtiter plates by hemagglutination. Characterization of the MoAbs was done by hemagglutination, immunoblotting, and epitope mapping.
Results: Eight immunoglobulin G MoAbs were identified. Five antibodies are specific by hemagglutination for s and two MoAbs, when diluted, are anti-S-like, but additional analyses shows a broad range of reactivity for GPB. Typing red blood cells (RBCs) for s from 35 donors was concordant with molecular analyses as were tests on RBCs with a positive direct antiglobulin test (DAT) from 15 patients. The anti-s MoAbs are most reactive with peptides containing the (31)QLVHRF(36) motif, with (29)Thr. By Pepscan analyses, the anti-S-like MoAbs reacted within the same regions as did anti-s, but independently of (29)Met. One antibody was defined serologically as anti-U; however, its epitope was identified as (21)ISSQT(25), a sequence common for both GPA and GPB.
Conclusion: In addition to their value as typing reagents, these MoAbs can be used to phenotype RBCs with a positive DAT without pre-test chemical modification.