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J Biol Chem, Vol. 274, Issue 36, 25862-25868, September 3, 1999
From the Goodpasture disease is a prototype autoimmune
disease characterized by the formation of autoantibodies against the
heterotrimeric basement membrane collagen type IV, which causes a
rapidly progressive glomerulonephritis. The pathogenic antibody
response is directed to the non-collagenous (NC1) domain of the The Goodpasture syndrome fulfills all criteria of a classic
autoimmune disorder in analogy to Koch's postulate for infectious diseases. The criteria demand that an adaptive immune response to a
self-antigen cause the observed pathology, as evidenced by transfer of
the disease in either a natural situation (for example, from mother to
child via the placenta) or an experimental disease model (via
autoantigen-specific antibodies or lymphocytes) (1). In Goodpasture
syndrome, the complex formation of autoantibodies with self-structures
in the glomerular basement membrane causes a rapidly progressive
glomerulonephritis, often accompanied by life-threatening pulmonary
hemorrhage. The self-epitope is localized to the NC1 domain of the The name Goodpasture syndrome was given in 1958 by Stanton and Tange
(4) in recognition of a patient described by Ernest Goodpasture in 1919 (5). In 1984, the antigen was identified as a new subunit of the NC1
domain of type IV collagen (6). This resulted in the cloning of the
antigen (7) and the designation of the Several studies aimed at identifying antibody epitopes of potential
pathogenic relevance have also been performed using experimental animal
models of autoimmune diseases. In contrast, little is known about
epitopes in human autoimmune diseases at a molecular level, most likely
due to methodological problems related to a diverse reactivity pattern
of the autoantibodies. More importantly, conformational requirements of
the epitopes may limit the application of linear synthetic peptides for
mapping strategies. Therefore, an experimental procedure was chosen
that allows the expression of the antigen as a recombinant protein in a
human cell line (HEK-293). This strategy enables the construction of a
variety of properly folded chimeric molecules in which the The present study focuses on the molecular characterization of the
Goodpasture epitope in the amino-terminal part of the Patients and Sera
The sera of 20 Goodpasture patients with biopsy-proven
anti-glomerular basement membrane nephritis were obtained from the serum bank at the Department of Nephrology, Lund University. All patients showed crescentic glomerulonephritis with linear deposits of
IgG in direct immunofluorescence. Seven of the patients also had an
overt lung hemorrhage. The mean age was 49 years (range, 18-78 years;
10 males and 10 females). The mean serum creatinine level at the time
of diagnosis was 920 µmol/liter, and 12 of the patients were
maintained on dialysis, 4 had died, and 4 survived with native
functional kidneys after 6 months of follow-up. Sera from 10 healthy
age-matched blood donors were used as controls.
Antibodies
Monoclonal antibodies against type X collagen (clone X53 (16))
as well as the previously characterized Mab17 monoclonal antibodies
raised against the Cell Culturing and Transfection
HEK-293 cells were cultured in 90-mm cell culture plates (Nunc,
Roskilde, Denmark) in Dulbecco's modified Eagle's medium:F-12 (1:1)
with 5% fetal calf serum (Life Technologies, Inc.). For each
construct, 5 µg of the linearized plasmid DNA was transfected into
1 × 106 HEK-293 cells using an electroporator
(Bio-Rad) with electrical settings of 200 V and 640 microfarads in a
0.4-cm cuvette. The transfected cells were seeded onto a new plate,
and, after 48 h, selection was started by supplementation with 800 µg/ml G418 (Life Technologies, Inc.). The medium was renewed every 2 days, and collection of the supernatants was started when the
G418-resistant cells reached confluence. During harvesting, the
transfected cells were kept in fetal calf serum-free Dulbecco's
modified Eagle's medium/F-12 supplemented with ascorbate (18).
DNA Constructs
All restriction enzymes and ligase were purchased from Roche
Molecular Biochemicals. The primers used are shown in Table
I. The Pfu-DNA polymerase was
purchased from Stratagene (La Jolla, CA).
Construction of Primary Vectors--
The primary type X/type IV
collagen chimera was constructed using Division of the NC1 Domain--
For the construction of six
Replacement Mutations of Single Amino Acid Residues in the
Replacement Mutations of Single Amino Acid Residues in the
General Handling of the Constructs--
After sequencing, all
constructs were tested for the translation of a protein with the
correct molecular weight using an in vitro system (Promega,
Madison, WI) with [35S]cysteine and T7 RNA polymerase.
Before transfection, the plasmid DNA was linearized using
ScaI.
ELISA
Native type IV collagen NC1 domains were purified as described
previously (10) and coated at 0.5 µg/ml. The coating efficiency of
the different recombinant proteins was calibrated by their equal
immunoreactivity with the anti-collagen type X monoclonal antibody
(16). Human sera were diluted 1:100, and the monoclonal antibodies were
diluted 1:1000. The ELISA was performed following standard procedures
(10) using alkaline phosphatase-conjugated swine anti-human IgG (Orion
Diagnostica AB, Trosa, Sweden) or rabbit anti-mouse IgG (DAKO,
Glostrup, Denmark) as secondary antibodies. Results were considered
positive when the absorbance values exceeded the mean + 2 S.D. of 10 healthy control sera. Notably, none of the control sera was positive in
any ELISA.
Inhibition ELISA
Dilutions of human sera were adjusted to give the same
absorbance after 1 h in a conventional ELISA with purified native
type IV collagen NC1 domains and preincubated overnight at 4 °C with the different inhibitors, i.e. recombinant or purified
native proteins in concentrations ranging from 0.0025 to 25 µg/ml.
The amount of IgG that reacted with surface-bound Goodpasture antigen (despite the presence of inhibitor molecules in the fluid phase) was
determined after extensive washing procedures with alkaline phosphatase-conjugated secondary antibodies as described above.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting
One ml of the cell supernatants was precipitated with Triton
X-100 and trichloroacetic acid. The precipitate was applied to SDS-polyacrylamide gel electrophoresis in 10-16% gradient gels under
nonreducing conditions (21). Immunoblot experiments were performed on
samples that were separated with SDS-polyacrylamide gel electrophoresis
and transferred to Immobilon polyvinylidene difluoride membrane
(Millipore, Saint-Quentin, France) (22).
For the construction of recombinant chimeric molecules that harbor
the Goodpasture epitope, the amino-terminal two-thirds of the
Goodpasture Disease
CHARACTERIZATION OF A SINGLE CONFORMATIONAL EPITOPE AS THE
TARGET OF PATHOGENIC AUTOANTIBODIES*
§,
Department of Nephrology, Lund University,
22185 Lund, Sweden, ¶ Department of Internal Medicine III,
University of Erlangen-Nuremberg, 91054 Erlangen, Germany, and
Wieslab AB, 22370 Lund, Sweden
ABSTRACT
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3
chain of type IV collagen (3(IV)NC1), but not to the homologous
region of the 1(IV)NC1. To identify the
conformation-dependent immunodominant epitope on the
3(IV)NC1, a variety of recombinant NC1 domains were constructed by
replacing single residues of 3(IV) with the corresponding amino
acids from the nonreactive 1(IV) chain. Replacement mutations were
identified that completely destroyed the Goodpasture epitope in the
3(IV) chain. Based on the identification of these critical
positions, the epitope was finally reconstructed within the frame of
the 1(IV) chain. The substitution of nine discontinuous positions in
the 1(IV)NC1 with amino acid residues from the 3 chain resulted
in a recombinant construct that was recognized by all patients' sera
(n = 20) but by none of the sera from healthy controls
(n = 10). This provides, for the first time, the
molecular characterization of a single immunodominant conformational
epitope recognized by pathogenic autoantibodies in a human autoimmune disease, representing the basis for the development of new
epitope-specific strategies in the treatment of Goodpasture disease.
INTRODUCTION
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3
chain of type IV collagen (3(IV)NC1), a major basement membrane
component of kidney glomeruli and pulmonary alveoli, thus explaining
the tissue distribution of the disease. The pathogenic role of the
B-cell response to this self-antigen is clearly supported not only by
the transfer of disease to monkeys using kidney-bound autoantibodies
from Goodpasture patients (2) but also by the therapeutic efficacy of
removal of circulating antibodies by plasma exchange, as well as that
of immunosuppressive treatments (3).
3(IV) chain. The epitope has
been characterized as a cryptotope because disintegration of the
supramolecular arrangement of the basement membrane type IV collagen by
denaturants leads to the exposure of critical epitope determinants,
thereby enhancing the immunoreactivity with patient sera (8). In turn,
chemical reduction completely abolishes immunoreactivity, indicating
the involvement of disulfide bonds in the stabilization of the epitope.
These features clearly indicate the involvement of a three-dimensional epitope that is normally hidden inside the intact glomerular basement membrane. Remarkably, despite an expected inter-patient variability, the ELISA1 reactivity of the
patients' autoantibodies has been successfully blocked with a
monoclonal antibody (9, 10), indicating a limited epitope recognition.
The carboxyl-terminal end of the 3(IV)NC1 domain has been suggested
to harbor the Goodpasture epitope, based on a study using synthetic
peptides (11). However, this study suffered from the disadvantage of
using linear peptides to characterize a three-dimensional epitope;
therefore, the results could not be subsequently confirmed. In turn,
more recent studies have pointed out the importance of the
amino-terminal region of the NC1 domain (12-14). Along this line, our
group has recently shown that only autoantibodies reacting with the
amino-terminal portion of the 3(IV)NC1 have a pathological impact on
kidney survival (15).
3(IV)NC1
sequence, which harbors the Goodpasture epitope, can be progressively
replaced by the corresponding sequence of the homologous 1(IV)NC1,
which is not recognized by the toxic autoantibodies. Indeed, the high
homology between both chains, as well as the conserved disulfide bonds, enhances the likelihood for proper folding of the hybrid constructs as
a prerequisite for conformation-dependent autoantibody binding.
3(IV)NC1 domain, to which the antibody response correlates with kidney disease.
For this purpose, chimeric constructs were devised in which the
clinically irrelevant central and carboxyl-terminal parts of the NC1
domain were expressed as 1(IV).
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3(IV)NC1 (17) were used for standardization of
the ELISAs and for immunoblotting experiments.
Primers used in this study
3(IV)NC1 cDNA cloned in
pBluescript SK
vector (Stratagene) with a BamHI site in
the 5' end and a NotI site in the 3' end. The full-length
type X collagen cDNA in the pBluescript SK vector was cloned
between the HindIII and NotI sites. Both plasmids
were cleaved with BamHI and NotI, and the
3(IV)NC1 cDNA was ligated into the internal BamHI
site of type X collagen cDNA. This construct contained five unique
restriction enzyme sites: a HindIII site in the 5' end of
the construct, a BamHI site in the junction between the type
X collagen and the type IV collagen NC1, HindII and
XbaI in the 3(IV)NC1 cDNA, and a NotI site
3' of the coding DNA. The BamHI, HindII,
XbaI, and NotI cleavage sites were introduced
into the 1(IV)NC1 cDNA by polymerase chain reaction
(Perkin-Elmer GeneAmp 2400) using primers 1-6. Upon restriction with
the appropriate enzymes, the fragments of the 1(IV)NC1 cDNA were
used to replace the corresponding cassettes of the wild type 3(IV)
sequence, as described previously (15). The constructs were restricted with HindIII and NotI and subcloned into a
cytomegalovirus promoter-driven expression vector (pcDNA3;
Invitrogene, Leek, the Netherlands). Because the major epitope region
could be localized to the amino-terminal domain of the 3(IV)NC1 (the
D1 construct in Table II), this region was subjected to further mutational analysis.
Construction design (see Fig. 1 for nomenclature and amino acid
positions)
1/3(IV) chimeric NC1 domains (the D2-D7 constructs in Table II),
the overlap extension polymerase chain reaction technique (19) was
applied using the primers 6-20 in Table I, using the D1 construct as a template.
3(IV) Sequence--
Fourteen positions were selected for mutational
analysis in a chimeric construct that harbors the amino-terminal
3(IV) NC1 (from A to G in Fig. 1). The selection criteria were based
on the identity in human and bovine 3(IV) sequences, as well as on
concurrent, nonconserved exchanges in the corresponding amino acid
residues of the human 1(IV) and 5(IV) sequences. These 14 amino
acid residues are emphasized in Fig. 1 by numbering their positions in
the sequence. The point mutations were introduced in the D1 construct
by the megaprimer method (20) using primers 6, 7, and 21-34.
1(IV) Sequence--
As the last step, replacement mutations were
introduced into chimeric constructs comprising the entire 1(IV)NC1
domain. By site-directed mutagenesis, five and nine codons were changed
from wild type 1(IV) to the corresponding 3(IV), respectively.
The substitutions were introduced by an overlap extension polymerase chain reaction (19) using primers 6 and 7 with primers 35-40 and
primers 6 and 7 with primers 15, 16, 19, 20, 39, and 40, respectively.
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1
chain of type X collagen, including the leader sequence, was fused to
the NC1 domain of type IV collagen (Fig.
1). The use of type X collagen in the
amino-terminal part of the recombinant molecule results in a protein
that is exported from the cells into the cell culture medium.
Furthermore, the type X collagen part of the molecule is invariable and
can therefore serve as a flag for protein detection. The epitope
mapping strategy with these constructs comprised four subsets of
experiments: 1) the NC1 domain was divided into three parts and
separately replaced the 3(IV) sequence with the corresponding
1(IV) sequence for each part. This was done in a previous study and
showed that the amino-terminal portion of the 3(IV)NC1 harbors the
major Goodpasture epitope and that only antibodies against this part of
the molecule correlate with kidney disease (15); 2) the immunoreactive
sequence was further narrowed down in the amino-terminal part of the
3(IV)NC1 domain; 3) point mutations were introduced in the
3(IV)NC1 to identify replacements that interfere with antibody
recognition; and 4) amino acid substitutions were performed in the
1(IV)NC1 at selected positions that reconstituted the Goodpasture
epitope in a domain that is normally not recognized by the patient
sera.
View larger version (13K):
[in a new window]
Fig. 1.
A, strategy for construction of the
basic chimera that was subsequently used as a basis for further epitope
mapping (for experimental details, see "Experimental Procedures"
and Table II). B, sequence comparison of the 3(IV) and
1(IV) chains in the amino-terminal part of the NC1 domain.
Letters indicate the borders of the constructs in the
regional mapping. Numbers indicate the positions selected
for replacement mutations (P1- P14 in Table II; for selection
criteria, see "Experimental Procedures").
Replacement of Subdomains of the Amino-Terminal Portion of the
3(IV)NC1 with the Corresponding 1(IV) Sequences--
To narrow
down the sequence within the D1 construct that harbors the major
epitope, six different constructs were generated that contained
replacements of different parts of the amino-terminal domain of
3(IV)NC1 with the corresponding sequences from the 1(IV). These
six chimeric proteins were designated D2-D7 (Table II). All sera
(n = 20) reacted with the D2, D3, and D5 chimeric proteins, whereas no antibody binding to D4 and D7 was detectable. A
subset of four sera also reacted with the D6 chimeric protein. Thus,
reactivity of the Goodpasture sera with the constructs indicated the
localization of the major epitope to a region consisting of 42 amino
acid residues between the positions C and F (as shown in Fig. 1).
However, for 20% of the sera, the requirements for antibody
recognition remained confined to the 15 amino acid residues of 3(IV)
from position C to E (Fig. 1).
Amino Acid Substitutions in the 3(IV) NC1 That Disturb
Autoantibody Recognition--
Comparison of the amino acid sequences
for the 1(IV) and 3(IV) chains revealed 33 nonconserved residues
in the amino-terminal portion of the NC1 domain from position A to G
(Fig. 1). For the identification of amino acid differences of critical
importance for antibody binding, the sequence comparison was extended
to bovine 3(IV) and human 5(IV) sequences. This strategy was
chosen because bovine 3(IV) is recognized by autoantibodies from
Goodpasture patients, in contrast to the nonreactive human 5(IV)
(23). Only positions with identity in the human and bovine 3(IV)
sequences but with differences from the corresponding human 1(IV)
and 5(IV) sequences were then selected for the introduction of
replacement mutations. The criterion was fulfilled by 14 amino acid
residues, the localization of which is indicated by the numbering of
positions 1-14 in Fig. 1. In these positions, the 3(IV)-specific
codons were replaced with the corresponding 1(IV) residues in
construct D1 (Table II), resulting in different chimeric molecules that harbor a single amino acid exchange (named P1-P14 in Table II). Despite slight differences in the reactivity of sera from different individuals with the recombinant proteins, two of the constructs, namely P9 and P10, remained totally negative with all patients' sera,
whereas the mutations P6, P7, and P13 at least partially abolished
their reactivity.
The results on the localization of critical positions for
immunoreactivity by single amino acid residue substitutions were in
concordance with the effect of replacement mutations of longer stretches of the 3(IV) sequence by the corresponding 1(IV)
domains in the amino-terminal portion of the NC1 domain. Thus, all
essential positions localize within the same 42-amino acid-long region
(between position C and F in Fig. 1) that has been shown in the
above-described experiments to require 3(IV) sequences for binding
of the Goodpasture sera. The reactivity of the four sera that remained
insensitive to the Q to R mutation in the P13 construct, in contrast to
the majority of Goodpasture samples (n = 16), had
previously been shown to be restricted to a subdomain of the critical
region (from position C to E in Fig. 1) that does not include the P13 position.
Reconstruction of the Goodpasture Epitope by Site-directed
Mutagenesis in the 1(IV)NC1--
Based on the knowledge of
positions in the 3(IV)NC1, in which a single replacement mutation
can destroy immunoreactivity, we attempted the reconstruction of the
Goodpasture epitope, within the frame of the nonreactive 1(IV)NC1,
by site-directed mutagenesis of five and nine amino acid residues,
respectively. First, the five critical residues in positions P6, P7,
P9, P10, and P13 (with a negative effect of mutations on autoantibody
binding) were replaced in the 1(IV)NC1 with the corresponding amino
acid residues I, S, P, S, and Q from the 3(IV) sequence (construct
S1 in Table II). Second, four additional nonconserved residues were
substituted that are localized in the region between position C and E
in Fig. 1. The resulting 1(IV)NC1 construct harbored eight amino
acid substitutions from the 3(IV) sequence between position C and E
in Fig. 1 and the amino acid exchange R to Q in the P13 position (construct S2 in Table II).
The first construct, S1, reacted only weakly with Goodpasture sera in
immunoblotting experiments (Fig. 2);
also, the mutational changes were insufficient to render the molecule
reactive with patient sera in ELISA. In contrast, the second chimeric
protein, S2, was recognized by all sera in both immunoblotting (Fig. 2) and ELISA, thereby indicating that the reactivity of the Goodpasture autoantibodies is directed toward this limited region in the NC1 domain
of type IV collagen. Notably, in this respect, there was no difference
between the epitope specificities of autoantibodies from patients with
or without overt lung bleeding. In contrast to the 20 Goodpasture
patients, there was no reactivity in the 10 sera of healthy humans used
as a control (Fig. 3). All reactivity of
the Goodpasture sera disappears upon reduction of the antigens (data
not shown).
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Affinity of Goodpasture Autoantibodies to the Chimeric
Constructs--
The affinity of the proteins to the Goodpasture sera
(n = 20) was determined by inhibition ELISA experiments
using recombinant 3(IV)NC1, D1, S1, and S2 to block the binding to
native NC1 domains from type IV collagen (Fig.
4). The results confirmed that the major
epitope in Goodpasture syndrome is preserved in the S2 chimeric protein, whereas the S1 construct proved insufficient for competitive binding, thus consistent with the results of a conventional ELISA shown
in Table II. Below saturation, the D1 and S2 chimeric proteins bound to
the Goodpasture antibodies with an affinity comparable to recombinant
3(IV)NC1. The maximal inhibitory capacity, in turn, differed
slightly between D1 and S2 on one side (approximately 85%, ranging
from 65% to 90%) and the complete 3(IV)NC1 (virtually 100%).
Thus, as many as 85% of the autoantibodies present in the Goodpasture
sera recognized an epitope that was fully contained in the critical
nine amino acid residues presented in the S2 construct (Table II).
Conversely, it was likewise remarkable that only 15% of the patient
autoantibodies recognized unrelated structures. These observations were
observed consistently in all 20 Goodpasture patients.
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ABSTRACT
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This study provides the first molecular characterization of a
conformational B-cell epitope that is the univocal target of pathogenic
autoantibodies in a human autoimmune disease. In Goodpasture syndrome,
the anti-type IV collagen autoantibodies cause immune complex formation
in the basement membranes of the kidney glomeruli and lung alveoli,
thereby leading to fatal kidney disease and lung bleeding. Because the
toxic autoantibodies are highly selective for the 3 chain of the
heterotrimeric type IV collagen, its immunoreactive domains were
subjected to a mutation analysis by replacements with the corresponding
sequences of the nonreactive, highly homologous 1(IV) chain in
chimeric recombinant constructs. Although the different sera showed
small differences in fine specificity, this mapping strategy allowed
the identification nine amino acid residues in the amino-terminal part
of the 3(IV)NC1 as the recognition site of all 20 sera derived from
biopsy-proven Goodpasture patients. Replacement mutations of these nine
amino acid residues from the 3(IV) into the wild-type 1(IV)
sequence converted the nonreactive 1(IV)NC1 into a recombinant
hybrid molecule that was recognized by all patients' sera with
comparable affinity to that of the native purified Goodpasture antigen,
as shown by competition ELISA. In contrast to most other autoimmune
diseases, 100% of the patients with Goodpasture disease have
autoantibodies against the 3(IV) chain. Furthermore, all Goodpasture
patients have one major epitope. In another autoimmune disease,
rheumatoid arthritis, antibodies against type II collagen are found in
30-70% of the samples (25). Furthermore, at least in animal models of
collagen II-induced arthritis, there seem to be antibodies against
several different epitopes (18), which complicates the epitope mapping considerably.
The identified nine amino acid residues are localized in the
amino-terminal part of the 3(IV)NC1 and form a discontinuous epitope
in close vicinity to the cysteine residues, which are critically
involved in the folding of the NC1 domain (24) (Fig. 5). The formation of disulfide bonds
between the cysteine residues stabilizes the tertiary structure in the
native NC1 domain, bringing the identified amino acid residues in a
reciprocal spatial relationship that is critical for antibody
recognition. Conversely, it is very likely that breakage of the
disulfide bonding dramatically affects the position of the critical
residues, consistent with earlier results documenting the loss of
immunoreactivity upon reduction of the Goodpasture antigen (8).
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The epitope localization within the complex suprastucture of the
basement membrane cannot be predicted at present, due to the lack of
appropriate structural models. Thus, the extent to which the critical
amino acid residues are normally exposed to the immune system and which
mechanisms enhance the accessibility for autoimmune recognition
in vivo remain a matter of speculation. Whereas the etiology
of Goodpasture syndrome is still unknown, the restricted reactivity of
autoantibodies from different patients strongly indicates an
antigen-driven autoimmune process. It is therefore tantalizing to
speculate that an immune response to a heterologous determinant of an
infectious agent may mimic the Goodpasture epitope in the
amino-terminal part of the 3(IV)NC1. The close correlation between
the specificity of the autoantibody response and the clinical outcome
indicates, in any case, that the epitope is part of a region with
important biological function, e.g. the maintenance of
basement membrane integrity. This may be disturbed by the interference
of antibody binding with the homotypic interactions of the collagen
molecules in the meshwork association.
The present investigation provides important structural information
about the immunodominant B-cell epitope in Goodpasture syndrome. This
is the first example in a human autoimmune disease of a
conformation-dependent epitope, which is recognized by the pathogenic autoantibodies in all affected individuals but by none of
the sera from healthy controls. Moreover, spreading of the autoantibody
response to other determinants seems to be a rather limited event in
Goodpasture disease. This new information may prove valuable not only
for diagnostic and prognostic purposes but also for the development of
therapeutic epitope-specific immunomodulatory strategies. The present
study may also bear important implications for the understanding of
other human autoimmune diseases.
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ACKNOWLEDGEMENTS |
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We thank Lena Gunnarsson for skillful technical assistance, Dr. I Girkontaite (Institute of Experimental Medicine, Erlangen, Germany) for the kind gift of anti-type X collagen, Dr. J Saus (FIBS, Valencia, Spain) for providing the cDNA clones encoding the type IV collagen, Prof. K. Von der Mark (Institute of Experimental Medicine, Erlangen, Germany) for the type X collagen cDNA, and Dr. E. Palombo-Kinne for reading the manuscript.
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FOOTNOTES |
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* This study was supported by Swedish Medical Research Council Grant 16X-09487, Riksförbundet för Njursjuka, the Crafoord Foundation, the Lundberg Foundation, the Magn. Bergvall Foundation, the Grönvall Foundation, the Wiberg Foundation, and the Deutsche Forschungsgemeinschaft (SFB 263, project C3).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Nephrology, Lund University Hospital, S-22185 Lund, Sweden. Tel.: 46-46-173541; Fax: 46-46-2114356; E-mail: Thomas.Hellmark@njur.lu.se.
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ABBREVIATIONS |
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The abbreviation used is: ELISA, enzyme-linked immunosorbent assay.
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) and 7 patients with lung hemorrhage (
)) and age-matched
healthy controls (n = 10) (
). ELISA
experiments showed a clear distinction between the Goodpasture patients
and the healthy control populations. The cut off value (the mean value
of the healthy controls + 2 S.D.) is indicated as a horizontal
line.
