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.