title: [Identification of the epitope of von Willebrand factor recognized by monoclonal antibody SZ-125 with immune-affinity mass spectrometry].
abstract: OBJECTIVE
  To identify the epitope of von Willebrand factor (vWF) recognized by monoclonal antibody SZ-125 (mAb SZ-125) using immune-affinity matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) assay in combination with peptide synthesis and amino acid site-mutagenesis technology.
  
  
  METHODS
  Recombinant vWF A3 domain (rVWF A3) was directly affinity bound to SZ-125 antibody beads and proselytized by trypsin. The digested peptide fragments were then measured using MALDI-TOF-MS. The detected peptide sequence by MALDI-TOF-MS was synthesized and several amino-acids in it were mutated to test its affinity with mAb SZ-125.
  
  
  RESULTS
  The epitope of rVWF A3 recognized by SZ-125 was identified to be the peptide fragment(1001);EGGPSQIGDALGFAVR(1016);. Synthesized peptide NH2;-EGGPSQIGDALGFAVR-COOH could bind to mAb SZ125.
  
  
  RESULTS
  of site-directed mutagenesis revealed that amino acids E1001, F1013, V1015 and R1016 played critical roles in the binding between mAb SZ-125 and rVWF A3.
  
  
  CONCLUSIONS
  The epitope of rVWF A3 recognized by mAb SZ-125 has been accurately confirmed using immune-affinity mass spectrometry in combination with peptide synthesis and site-directed mutagenesis of special amino acids.
authors: Jiangsu Institute of Hematology, Suzhou, China.
authors: Xuemei
authors: X
authors: Li
authors: Fuqiang
authors: F
authors: Wang
authors: Fei
authors: F
authors: Shen
authors: Yiming
authors: Y
authors: Zhao
authors: Miao
authors: M
authors: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D018604:Epitope Mapping / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D014357:Trypsin