Production of human monoclonal anti-Jk3, recognising an epitope including the Jk(a) /Jk(b) polymorphic site of the Kidd glycoprotein.

BACKGROUND AND OBJECTIVES: The Kidd blood group system consists of polymorphic antigens, Jk(a) (JK1) and Jk(b) (JK2), and a high-incidence antigen, Jk3. Anti-Jk3 is often observed in immunised Jk(a-b-) individuals. In this study, we aimed to establish a human hybridoma cell line secreting monoclonal anti-Jk3 (HIRO-294). MATERIALS AND METHODS: Peripheral blood lymphocytes of a Filipino woman with the Jk(a-b-) phenotype having anti-Jk3 were transformed with Epstein-Barr virus and then hybridised with the myeloma cell line JMS-3 using the polyethylene glycol (PEG) method. The reactivity and specificity of the anti-Jk3 were examined by serology and flow cytometry. RESULTS: Four hybridoma clones secreting anti-Jk3 were established and the antibody from one of these clones, HIRO-294, was examined. The reactivity of HIRO-294 was positive with 227 Jk(a+b-) red blood cells (RBCs), 298 Jk(a-b+) RBCs, and 1043 Jk(a+b+) RBCs, but was negative with 21 Jk(a-b-) RBCs. Eluates from Jk(a+b-) RBCs and Jk(a-b+) RBCs sensitised with the anti-Jk3 were cross-reacted with Jk(a-b+) RBCs and Jk(a+b-) RBCs, respectively. The reactivity of HIRO-294 was enhanced by the treatment of RBCs with ficin, trypsin, pronase and α-chymotrypsin, but was not changed by their treatment with neuraminidase, dithiothreitol and ethylenediaminetetraacetic acid (EDTA) glycine acid (GA). The RBCs sensitised by the anti-Jk3 were not agglutinated with the commercial reagents of anti-Jk(a) and anti-Jk(b) by saline test, whereas the nonsensitised RBCs or those sensitised by monoclonal anti-D [HIRO-3, immunoglobulin G (IgG) class] were agglutinated with those reagents. CONCLUSIONS: We established a human hybridoma cell line secreting monoclonal anti-Jk3 (HIRO-294). This antibody had unique specificity, recognising the Kidd glycoprotein including the Jk(a) /Jk(b) polymorphic site.

Presence of nucleotide substitutions in transcriptional regulatory elements such as the erythroid cell-specific enhancer-like element and the ABO promoter in individuals with phenotypes A3 and B3, respectively.

BACKGROUND AND OBJECTIVES: An erythroid cell-specific regulatory element, referred to as the +5.8-kb site, has been identified in the first intron of the human ABO blood group gene. Subsequent studies have revealed involvement of deletion or mutation at the site in phenotypes Am , Bm and ABm . We investigated the molecular mechanisms involved in the A3 and B3 phenotypes. MATERIALS AND METHODS: Genomic DNAs were prepared from peripheral blood of seven A3 individuals and twelve B3 or AB3 individuals, and the nucleotide sequences were investigated using PCR and sequencing. Promoter assays were performed with K562 cells. RESULTS: Two single point-mutations at +5893 or +5909 in the site on the A-allele were found in A3 individuals, while promoter assays revealed decreased activity at the site as a result of each substitution. In two B3 individuals, a single point-mutation at -77 in the ABO promoter on the B-allele was found, and the substitution was demonstrated to reduce the promoter activity. CONCLUSION: Nucleotide substitutions in the transcriptional regulatory elements such as the +5.8-kb site and the ABO promoter appear to decrease transcription from the A- and B-alleles, resulting in reduction in A- and B-antigen expression in A3 and B3, respectively.