title: Prokaryotic versus eukaryotic recombinant Lutheran blood group protein for antibody identification.
abstract: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Seltsam
authors.forename: Daniela
authors.initials: D
authors.lastname: Grüger
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
mesh_terms: D000906:Antibodies / Q000032:analysis* / Q000276:immunology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells; D015870:Gene Expression; D006388:Hemagglutinins / Q000276:immunology; D006801:Humans; D008188:Lutheran Blood-Group System / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011387:Prokaryotic Cells; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000906:Antibodies; D006388:Hemagglutinins; D008188:Lutheran Blood-Group System; D011994:Recombinant Proteins