Sequential analysis by immunoprecipitation-MALDI-TOF: a novel method for detection and identification of alloantibody specificities.
Alloantibodies are known to influence transplant outcomes. Apart from human leukocyte antigens (HLA), non-HLA targets have been suggested to play a significant role, but little is known about their nature. Here, we present a novel method for identification and characterization of cell surface antigens bound by alloreactive antibodies. Our method consists of 2 consecutive steps: first, immunoprecipitation of cell surface proteins is carried out with serum and, second, matrix-assisted laser desorption/ionization-time-of-flight is used to fingerprint the precipitated cell-surface proteins. As an example, we performed immunoprecipitation with peripheral blood lymphocytes, which had been incubated with an alloreactive serum; immune complexes were coupled to protein-G beads and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; differential protein fractions were then analyzed by matrix-assisted laser desorption/ionization-time-of-flight. The method was validated with serum as well as with plasmapheresis material, which contained antibodies of known HLA specificities, demonstrating its applicability for clinical use.
Department of Transplantation Immunology, Institute of Immunology, University of Heidelberg, Heidelberg, Germany.
Andreas
A
Heinold
Boris
B
Kuehl
Gerald
G
Brenner-Weiss
Gerhard
G
Opelz
Thuong Hien
TH
Tran
D000595:Amino Acid Sequence; D000918:Antibody Specificity / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006680:HLA Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D007518:Isoantibodies / Q000276:immunology*; D007519:Isoantigens / Q000276:immunology; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D006680:HLA Antigens; D007518:Isoantibodies; D007519:Isoantigens