4 research outputs found
Comparison of the established standard complement-dependent cytotoxicity and flow cytometric crossmatch assays with a novel ELISA-based HLA crossmatch procedure
The detection of donor-specific anti-HLA
antibodies by standard procedures such as complementdependent
cytotoxicity assay (CDC) or flow cytometric
(FACS) analysis is limited by its low sensitivity and the
quality of the donor cells. Therefore, an ELISA-based
technique was employed using solid phase-immobilized
monoclonal antibodies to capture HLA class I or class II
molecules of the donor, respectively. In this HLA class I
and class II antibody monitoring system (AMS) the
donor-specific anti-HLA antibodies from the sera of
recipients bind to the HLA molecules of the donor which
have been immobilized by monoclonal antibodies (mAb)
recognizing non-polymorphic epitopes. Upon binding of
donor-specific anti-HLA antibodies they are recognized
by secondary enzyme-conjugated anti-human
immunoglobulin (Ig) antibodies. A newly established
modification of the standard protocol allows the
differentiation between bound antibodies of the IgG and
IgM isotype. Furthermore, this assay was adapted for
investigating small amounts of solid tissue of donors
from whom no other cells (e.g. from blood) were
available. We here provide an overview of the classical
crossmatch methods with their advantages and limits. In
addition, the design of the novel AMS-ELISA is
described in terms of quality and sensitivity of the
approach using exemplary cases of different application.
The selected cases show that the AMS-ELISA represents
a valuable tool for the post-transplantation monitoring of
donor-specific anti-HLA antibodies during reaction
crisis, after transfusion reactions and in particular cases
of tissue transplantations lacking single cells
Soluble CD30 serum level - an adequate marker for allograft rejection of solid organs?
The CD30 molecule, a 120 kDa cell surface
glycoprotein, is a member of the tumor necrosis factor
receptor (TNF-R) superfamily and was originally
identified on the surface of Reed-Sternberg cells and
anaplastic large cell lymphomas in Hodgkin’s disease
patients. In addition to lymphoproliferative disorders the
expression of CD30 was found in both activated CD8+
and CD4+ Th2 cells which lead to the activation of Bcells
and consequently to the inhibition of the Th1-type
cellular immunity. The membrane-bound CD30
molecule can be proteolytically cleaved, thereby
generating a soluble form (sCD30) of about 85 kDa.
Low serum levels of soluble CD30 were found in
healthy humans, whereas increased sCD30 serum
concentrations were detected under pathophysiological
situations such as systemic lupus erythematosus,
rheumatoid arthritis, certain viral infections and adult T
cell leukaemia/lymphoma. In addition, it has recently
been suggested that pre- or post-transplant levels of
sCD30 represent a biomarker for graft rejection
associated with an impaired outcome for transplanted
patients. We here review (i) the current knowledge of the
clinical significance of sCD30 serum levels for solid
organ transplantations and (ii) our own novel data
regarding inter- and intra-individual variations as well as
time-dependent alterations of sCD30 levels in patients. (iii) Based on this information the implementation of
sCD30 as predictive pre-transplant or post-transplant
parameter for solid organ transplantation is critically
discussed
Recombinant generation of two fragments of the rat complement inhibitory factor H [FH(SCR1-7) and FH(SCR1-4)] and their structural and functional characterization in comparison to FH isolated from rat serum
Factor H (FH) is the predominant soluble
inhibitor of the complement system. With a
concentration of 200-800 µg/ml in human and rat plasma
it acts as a cofactor for the soluble factor I (FI)-mediated
cleavage of the component C3b to iC3b. Furthermore it
competes with factor B for binding to C3b and C3(H2O)
and promotes the dissociation of the C3bBb complex.
FH is a monomer of about 155 kDa which comprises 20
short consensus repeats (SCR), each of which is
composed of approximately 60 amino acid (aa) residues.
Two functional fragments of FH comprising the SCR1-4
or SCR1-7 were generated using either the Baculovirus
system or stably transfected human embryonal kidney
cells, respectively. These fragments, as well as FH
purified from rat serum, were first analyzed for their
relative molecular weights (Mr) using non-reducing or
reducing SDS-PAGE. The Mr of the FH variants differed
by about 20 % depending on the experimental conditions
employed. Only the Mr of proteins separated under
reducing conditions were in accordance with the MW
calculated from the aa sequence. Analyses of the
glycosylation patterns using PAS-staining showed a lack
of staining of the recombinant variants (SCR1-4 and
SCR1-7) in contrast to FH(SCR1-20) from serum. Using
a complement hemolysis assay (CH50-assay) all three
variants exhibited a molar complement inhibitory
activity of FH(1-20)/FH(1-7)/FH(1-4) of about 3/1/1. These data support the postulated model of FH bearing
three binding sites for its ligand C3b, from which one is
located in the SCR1-4, whereas the other two are located
in the SCR8-20