ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Glycophorins/immunology , Immunization , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/classification , Glycophorins/chemistry , Hemagglutinins/analysis , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptide Fragments/chemistrySubject(s)
Blood Donors , Blood Group Antigens/genetics , Hispanic or Latino/genetics , Humans , PhenotypeABSTRACT
BACKGROUND: Some low-incidence antigens in the Rh blood group system (e.g., VS, Rh32, FPTT) are expressed by more than one Rh complex. We describe a new low-incidence antigen that is present on RBCs with the partial D phenotypes, DIIIa or DOL, on RN RBCs and on one example of STEM+S RBCs. STUDY DESIGN AND METHODS: Standard hemagglutination testing was performed with two sera that agglutinated DIIIa RBCs on our in-house antibody identification panel. DNA-based assays were performed on selected samples. RESULTS: RBCs with the DIIIa (n = 31), DOL (n = 5), or RN (n = 10) phenotype were agglutinated by both sera, as were RBCs from one STEM+S person. Reactivity with RBCs of either DIIIa or DOL phenotypes was stronger than with RN RBCs and could not be separated by adsorption and elution. CONCLUSION: An antibody, anti-DAK, which recognizes a novel low-incidence antigen that is more strongly expressed on DIIIa and DOL RBCs than on RN RBCs is described. The antibody agglutinated RBCs from 4 percent of D+ African American blood donors in New York. The antigen, DAK, has been assigned the ISBT number RH54 (004.054).
