Abstract:

O24

SEQUENTIAL ANALYSIS BY IMMUNOPRECIPITATION-MALDI-TOF

MASS SPECTROMETRY FOR DETECTION AND IDENTIFICATION OF

ALLOANTIBODY SPECIFICITIES

Andreas Heinold1, Boris Kühl2, Gerald Brenner-Weiss2, Gerhard Opelz1,

Thoung H. Tran1

1Immunologie, Heidelberg, Germany; 2Karlsruher Institut für Technologie, Karlsruhe, Germany

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 two consecutive steps: first,

immunoprecipitation of cell surface proteins is carried out with a serum or

plasma; second, matrix-assisted laser desorption/ionization-time of flight

(MALDI-TOF) mass spectrometry (ms) is employed to fingerprint the precipitated

cell surface proteins. The method has been designated as SIMT for Sequential

analysis of Immunoprecipitation followed by MALDI-TOF ms. As an example,

we performed immunoprecipitation with peripheral blood lymphocytes which had

been incubated with an anti-HLA-B27 serum; immune complexes were coupled

to protein G beads and separated by SDS-PAGE; differential protein fractions

were then analyzed by MALDI-TOF ms. After immunoprecipitation of lymphocytes

with an anti-HLA-B27 serum and SDS-PAGE, a protein band (40 kDa)

was clearly observed in the precipitate of HLA-B27-positive cells, but was absent

in the precipitate of HLA-B27-negative cells. The protein of the 40 kDa band

was subsequently identified by MALDI-TOF ms as HLA-B27. Apart from using

sera, the method was also validated with plasmapheresis material which

contained antibodies of known HLA specificities. Unlike serum, which is usually

available only in limited amounts, plasma collected during plasmapheresis

(approximately 4 liters) can be used for extensive laboratory testing. SIMT

proved to be a novel method which can be used in clinical settings to detect and

to specify the targets of alloantibodies. In principle, it may enable the

identification of hitherto unknown alloantibody specificities. Compared to other

methods used for the characterization of unknown antigens (e.g. SEREX and

protein arrays), SIMT is specific for genuine cell surface proteins. Furthermore,

we show here that the SIMT method can be successfully performed with plasma

collected during plasmapheresis.