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.
Heinold
Andreas
A
Department of Transplantation Immunology, Institute of Immunology, University of Heidelberg, Heidelberg, Germany.
Kuehl
Boris
B
Brenner-Weiss
Gerald
G
Opelz
Gerhard
G
Tran
Thuong Hien
TH
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