title: Application of α-N-acetylgalactosaminidase and α-galactosidase in AB to O red blood cells conversion.
abstract: BACKGROUND
  Enzymatical conversion of A or B RBCs into group O RBCs (ECORBCs) was achieved by using α-N-acetylgalactosaminidase and α-galactosidase, respectively. Now, we initiated AB to O-RBC conversion by using these two enzymes together. But α-N-acetylgalactosaminidase and α-galactosidase's preserving and their reaction buffer were quite different. The aim of this study is to confirm an available system for converting AB to O RBCs, especially to study the maximal permission amount of PCS which was brought to the system-accompanied enzyme addition.
  
  
  METHODS
  Enzyme activity was detected by using GalNAc-pNp or Gal-pNp as substrates. The efficiency of the conversion of A or B antigen was evaluated by routine method and measured by fluorescence-activated cell sorting analysis. The optimal buffer component and the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed according to A and B antigen epitope removal efficiency.
  
  
  RESULTS
  The activity of α-N-acetylgalactosaminidase and α-galactosidase was not decreased drastically when they were kept in PCS Buffer in 4°C. The optimal reaction buffer composed of glycine 250 mM and NaCl 3 mM, pH 6.8 and PCS less than 10%(v/v). For converting A(1)B to O RBCs completely, the doses of α-N-acetylgalactosaminidase and α-galactosidase were confirmed as 0.015 mg/ml packed RBCs(pRBCs) for A(1) antigen epitopes and 0.005 mg/ml pRBCs for B epitopes. Approximately 0.004 mg α-N-acetylgalactosaminidase and 0.005 mg α-galactosidase were required to convert 1 ml pRBCs.
  
  
  CONCLUSIONS
  Our studies indicated that α-N-acetylgalactosaminidase and α-galactosidase were stable in PCS buffer and a modified protocol which was propitious to converting AB to O RBCs was provided.
authors.affiliation: Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine, Beijing, PR China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Gao
authors.forename: Subo
authors.initials: S
authors.lastname: Li
authors.forename: Yingxia
authors.initials: Y
authors.lastname: Tan
authors.forename: Shouping
authors.initials: S
authors.lastname: Ji
authors.forename: Yingli
authors.initials: Y
authors.lastname: Wang
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Bao
authors.forename: Lijuan
authors.initials: L
authors.lastname: Xu
authors.forename: Feng
authors.initials: F
authors.lastname: Gong
mesh_terms: D000017:ABO Blood-Group System / Q000737:chemistry / Q000378:metabolism*; D001803:Blood Transfusion / Q000379:methods*; D002021:Buffers; D002469:Cell Separation; D004912:Erythrocytes / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D055550:Protein Stability; D000519:alpha-Galactosidase / Q000737:chemistry / Q000378:metabolism*; D048809:alpha-N-Acetylgalactosaminidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000017:ABO Blood-Group System; D002021:Buffers; D000519:alpha-Galactosidase; D048809:alpha-N-Acetylgalactosaminidase