Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

Aberrant intracellular trafficking of a variant B glycosyltransferase.

BACKGROUND: Little is known about the mechanism by which amino acid polymorphisms outside the catalytically active cleft of ABO glycosyltransferases cause weak ABO phenotypes. STUDY DESIGN AND METHODS: Extensive ABO phenotyping and genotyping were performed to classify the blood of a healthy blood group O donor with weak isoagglutinins. ABO antigen and glycosyltransferase expression profiles were then studied in eukaryotic transfection experiments, and the topology of ABO glycosyltransferase was analyzed. RESULTS: The donor's red blood cells were retyped as A(weak), and his serum contained weakly reactive anti-A and anti-B. Sequence analysis revealed two novel ABO alleles. A donor splice-site mutation detected at the exon 6/intron 6 junction of an ABO*A101 allele was predicted to result in skipping exon 6 in the mRNA. The other haplotype displayed a single 688G>C substitution predicting a Gly230Arg exchange in the catalytic domain in an otherwise normal ABO*B101 allele. The transfection studies revealed very weak expression of B antigen by the novel ABO*B allele. According to the topologic analysis, steric hindrance due to the Gly230Arg exchange may cause conformational changes in the variant B transferase. Compared to the wild-type B transferase, the transfected cells exhibited lower-level protein expression and intracellular dislocation. CONCLUSION: This study provides first evidence that aberrant trafficking of variant ABO transferases may be involved in the formation of weak ABO phenotypes.

Weak blood group B phenotypes may be caused by variations in the CCAAT-binding factor/NF-Y enhancer region of the ABO gene.

BACKGROUND: Binding of CCAAT-binding factor NF-Y (CBF/NF-Y) to a 43-bp repeat unit in the minisatellite region in the 5' region of the ABO gene (CBF/NF-Y enhancer region) plays an important role in regulating the transcription of ABO genes. The common ABO alleles were found to have CBF/NF-Y enhancer regions with specific numbers of 43-bp minisatellite repeats. MATERIAL AND METHODS: Blood samples from four healthy blood donors with weak B phenotypes were subjected to extensive ABO genotyping, including nucleotide sequencing of the 5' regulatory region containing the CBF/NF-Y enhancer. RESULTS: The coding region of the ABO genes exhibited common ABO*B101-heterozygous genotypes in all samples, but unexpected variations were observed in the CBF/NF-Y enhancer region. In two cases, the CBF/NF-Y enhancer motifs did not exhibit the expected ABO allele dependency. One, an AB(weak) sample was heterozygous for ABO*A101 and ABO*B101 but homozygous for the ABO*B101-specific CBF/NF-Y motif. The second had a common ABO*B101/ABO*O01 genotype but was heterozygous for ABO*A101- and ABO*O01-specific enhancer motifs. In the other two samples, novel CBF/NF-Y motifs were found. One contained a shortened version of an otherwise ABO*B101-specific CBF/NF-Y motif, and the other had a single-base substitution located 12 bp upstream from the beginning of the first 43-bp repeat of an ABO*B101-specific CBF/NF-Y enhancer sequence. CONCLUSION: The frequency of variations in the CBF/NF-Y region of the ABO gene in these samples with presumably common ABO*B101 alleles suggests that weak blood group B phenotypes may be caused by sequence variations in the CBF/NF-Y regulatory region.

Prokaryotic versus eukaryotic recombinant Lutheran blood group protein for antibody identification.

BACKGROUND: At present, identification of antibodies against high-frequency antigens is limited to reference laboratories having panels of rare red blood cell (RBC) specimens in stock. Antibodies against Lu(b) are among the most frequent clinically relevant antibody specificities directed against high-frequency antigens. STUDY DESIGN AND METHODS: Soluble recombinant Lu(b) fusion proteins consisting of the first three N-terminal immunoglobulin superfamily domains and a V5-His tag were generated. Eukaryotic recombinant Lu(b) proteins were isolated from cell culture supernatant of stably transfected HEK293 cells with anti-V5 Sepharose. Prokaryotic Lu(b) fusion proteins were expressed in Escherichia coli, purified by Ni-NTA, and refolded by chromatographic procedures. Ten anti-Lu(b) serum samples, 6 anti-Lu(a) serum samples, 30 serum samples directed against other blood group antigens, 10 serum samples from patients with RBC autoantibodies, and 100 serum samples from randomly selected donors were used for antibody screening. RESULTS: Eukaryotic and prokaryotic recombinant Lu(b) proteins proved to be equally suited for identification of anti-Lu(b). Recombinant Lu(b) protein-based enzyme-linked immunosorbent assay correctly identified samples containing anti-Lu(b) sera, and the titers were at least two times higher than those measured by the gel agglutination-based indirect antiglobulin test. In hemagglutination inhibition assays, recombinant Lu(b) protein neutralized all anti-Lu(b), but none of the other alloantibodies decreased in reactivity. CONCLUSION: Antibody detection systems based on soluble eukaryotic or prokaryotic recombinant blood group proteins have the potential to replace current systems with rare RBCs for identification of alloantibodies against high- or low-frequency antigens. This innovation could bring routine laboratories one step closer to specialized antibody diagnostics.