close

Вход

Забыли?

вход по аккаунту

?

Human autoantibody secreted by immortalized lymphocyte cell line against the 68k polypeptide of the u1 small nuclear ribonucleoprotein.

код для вставкиСкачать
1265
HUMAN AUTOANTIBODY SECRETED BY
IMMORTALIZED LYMPHOCYTE CELL LINE AGAINST
THE 68K POLYPEPTIDE OF THE
U1 SMALL NUCLEAR RIBONUCLEOPROTEIN
JINTAO CHEN, YOSHIHIKO TAKEDA, W. ERIC VANDERSLICE, GORDON C. SHARP,
INGVAR PETTERSSON, ANDERS ROSEN, HANS WIGZELL, and RICHARD J . WANG
Autoantibody of an immortalized human lymphocyte cell line, Su-2E4, derived from peripheral lymphocytes of a patient with mixed connective tissue
disease, showed specific binding of the 68K polypeptide
of U1 small nuclear RNP (snRNP) and immunoprecipitation of U1 RNA. The reaction patterns of Su-2E4 and
a murine monoclonal anti-(U1)snRNP line, 2.73, and
results of a competition assay with the 2 antibodies
suggest similar, but not necessarily identical, epitope
recognition.
Autoantibodies to nuclear and cytoplasmic
components are frequently found in patients with
systemic rheumatic diseases (1-3). The autoantigens
are often nucleic acid-associated proteins, such as
small nuclear RNP (snRNP) (4). Circulating autoantibodies to some of these antigens are characteristic of
Presented by Jintao Chen in partial fulfillment of the requirements for a PhD degree, University of Missouri, Columbia.
From the Dalton Research Center, Division of Biological
Sciences, and the Department of Medicine, University of Missouri,
Columbia, and the Departments of Immunology and Tumor Biology,
Karolinska Institute, Stockholm, Sweden.
Supported by NIH grant AR-20305. Mr. Vanderslice's
work was supported by an NIH predoctoral training grant.
Jintao Chen, BS: Division of Biological Sciences, University of Missouri: Yoshihiko Takeda, MD: Department of Medicine,
University of Missouri: W. Eric Vanderslice, BS: Division of
Biological Sciences, University of Missouri: Gordon C. Sharp, MU:
Department of Medicine, University of Missouri: lngvar Pettersson,
PhD: Department of Immunology, Karolinska Institute: Anders
Rosin, MD: Department of Tumor Biology, Karolinska Institute;
Hans Wigzell, MD: Department of Immunology, Karolinska Institute: Richard J . Wang, PhD: Dalton Research Center, Division of
Biological Sciences, University of Missouri.
Address reprint requests to Richard J. Wang, PhD, Dalton
Research Center, University of Missouri, Columbia, MO 6521 I .
Submitted for publication February IS, 1988: accepted in
revised form May 23, 1988.
Arthritis and Rheumatism, Vol. 31, No. 10 (October 1988)
certain diseases. For example, anti-Sm antibodies are
strongly associated with systemic lupus erythematosus (SLE), and high titers of antibodies against
nuclear R N P are frequently present in the sera of
patients with mixed connective tissue disease (MCTD)
(5-7).
Small nuclear RNP are important components
in pre-messenger RNA processing (8,9). They consist
of at least 1 1 polypeptides (termed 68K, A, A', B, B',
B", C, D, E, F, and G), over a molecular weight range
of 11-70 kd, and 5 snRNA molecules (UI, U2, U4, US,
and U6) (10-13). Human serum autoantibodies to
snRNP are able to precipitate some or all of the U
snRNA (14). Anti-Sm antibodies precipitate U1, U2,
U4, U5, and U6, and recognize primarily B/B' and D.
Antibodies against (U 1)snRNP precipitate only U1
and recognize UI-specific polypeptides 68K, A, and
C. Anti-(U I ,U2)snRNP autoantibodies precipitate
both U1 and U2 and recognize UZspecific polypeptides A' and B" (15). Recent immunoblotting analyses
and enzyme-linked immunosorbent assays (ELISA)
using isolated ( U 1)snRNP polypeptides have revealed
a strong association of autoantibodies to 68K with
MCTD (16,17).
In the elucidation of the biochemical structure
and possible biologic and autoantigenic roles of the
snRNP particles, difficulties are frequently encountered in using autoimmune patient sera as probes,
because they frequently contain multiple autoantibody
specificities. Use of the technologies of hybridoma (18)
and Epstein-Barr virus (EBV) transformation (19-2 1)
has facilitated understanding and evaluation of autoimmune disorders (22-25). Murine monoclonal antibodies (MAb) against snRNP have proved to be very
useful specific probes, with several isolated against
CHEN ET AL
1266
(U1)snRNA-specific polypeptides (26-29). For study
of human autoimmunity and immune regulation and
for application in clinical diagnosis and treatment of
human autoimmune diseases, however, use of human
autoantibodies produced by immortalized human cell
lines is desirable. We are not aware of any previous
report on such human autoantibodies produced in
culture against the 68K polypeptide. Here we report
the isolation and characterization of a human autoantibody against the U 1-specific 68K polypeptide,
generated by EBV transformation of lymphocytes
from a patient with MCTD.
MATERIALS AND METHODS
Establishment of human antibody-producing lines.
EBV-transformed human lymphocytes were prepared as
described previously (19,2 1). Briefly, human peripheral lymphocytes from a patient with MCTD were separated on a
Histopaque gradient (Sigma, St. Louis, MO) and stimulated
with 1% pokeweed mitogen (Gibco, Grand Island, NY). For
24 hours, 1.5 X lo7 cells were incubated with 10 ml of
prefiltered EBV-containing culture supernatant from B95-8
cells. The infected lymphocytes were then collected, washed
with RPMI 1640 medium, and cultured in RPMI 1640 medium containing 20% fetal bovine serum (FBS) at a cell
density of 5 x 104/0.2-ml well. Culture supernatants were
screened by ELISA as described elsewhere (30), using
extractable nuclear antigen (ENA) (5) as the antigen. Positive cultures were cloned by limiting dilution.
Murine monoclonal anti-(U 1)snRNP line 2.73 (26)
was kindly supplied by Dr. Sallie 0. Hoch. Murine monoclonal anti-Sm line Y12 (31) and the 2.73 line were grown in
RPMI medium with FBS.
Purification of (U1)snRNP antigen. Rabbit thymus
extract (32) was applied to an anti-(U 1)snRNP affinity column, prepared by coupling total IgG isolated from an MCTD
anti-(U1)snRNP serum to cyanogen bromide-activated
Sepharose 4B (Pharmacia, Piscataway, NJ). The column
was then washed with 10 mM phosphate buffer (pH 7.5)
containing 0.5M NaCl and 0.2 mM phenylmethylsulfonyl
fluoride (PMSF; Sigma), and the bound protein was eluted
with 3M NaSCN. The eluate was dialyzed against the
column washing buffer and concentrated to 1.5 mg protein/ml by ultrafiltration. Polypeptides 68K, A, B/B', C, and
D were identified in the resulting (U 1)snRNP preparations
by protein staining and immunoblotting analysis (26).
Preparation of (U1)snRNP polypeptides. Purified
(U1)snRNP was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (33), in which 3
mm-thick 11% gels were loaded with the snRNP at 1.5 mg
proteidgel. Peripheral vertical slices of each gel were cut
and stained with Coomassie brilliant blue. Unstained gel
bands corresponding to individual (U 1)snRNP polypeptides
were excised, homogenized in 10 mM phosphate buffered
saline (PBS; pH 7.4), and centrifuged at 10,OOOg for 1 hour.
The supernatants were then collected as snRNP polypeptide
preparations.
Immunoblotting. Immunoblotting was performed as
previously described (34), except that purified (U1)snRNP
was loaded onto 13% SDS-polyacrylamide gels at 0.012 mg
proteinkm of gel slot.
ELISA using (U1)snRNP polypeptides. For the polypeptide ELISA, polyvinyl microtiter plates (Dynatech, Alexandria, VA) were incubated overnight at 4°C with individual snRNP polypeptides, and then for 3 hours at room
temperature with 3% bovine serum albumin (BSA) in PBS
with 0.2 mM PMSF. Culture supernatants, diluted when
necessary with culture medium containing 15% FBS, were
added to the wells and incubated overnight at 4°C. Appropriate horseradish peroxidase-conjugated secondary antibodies (Accurate, Westbury, NY), diluted to 1:1,00&1:2,500
in PBS-Tween 20 (0.05% Tween 20 in PBS), 1% BSA, and
1% bovine gamma globulin, were incubated in the wells for
3 hours at room temperature. Subsequently, 0.055% ophenylenediamine and 0.01% H202 in 0.1M citrate buffer
(pH 5.0) were added, and color development was measured
at 450 nm.
Competitive (U1)snRNP polypeptide ELISA. For
competition between polypeptide antigens, purified polypeptides were dialyzed against PBS and incubated overnight at
4°C with dilutions of MAb supernatants at a I :I ratio. The
antigen-antibody mixtures were then added to microtiter
plates coated with appropriate polypeptide antigens. The
remaining steps were performed as described for the snRNP
polypeptide ELISA.
To detect competition between different antibodies,
the addition of an antibody to an antigen-coated microtiter
plate was preceded by reacting a competing antibody in the
plate overnight at 4°C. Subsequent steps were performed as
described above.
RNA immunoprecipitation. Precipitation of U RNAs
with anti-snRNP IgG antibodies was performed as described
(14,16,35). For precipitation with IgM antibodies, the foflowing modifications (Vanderslice WE, Wang RJ: unpublished
observations) were used. Protein A-Sepharose beads (0.05
ml; Repligen, Cambridge, MA) were incubated for 15 hours
at 4°C with 0.2 mg of goat anti-human IgM antibody (Kallestad, Austin, TX) as intermediate antibody. The antiIgM-charged beads were then used for RNA precipitation as
cited above.
RESULTS
An immortalized human lymphocyte cell line,
designated Su-2E4, was isolated after EBV transformation of peripheral blood lymphocytes from a patient
with MCTD. This line was recognized as an antibodyproducing culture by ELISA screening with ENA as
the antigen, and was found to produce human IgM
(data not shown).
Anti-68K immunoblotting activity. A weak binding activity of Su-2E4 against a polypeptide of MW
68K was initially revealed by immunoblotting with
human Jurkat cell extract (12) as the antigen (data not
shown), in addition to a 60K polypeptide and a 120K
ANTI-68K POLYPEPTIDE REACTIVITY
1267
polypeptide, probably due to cross-reactivity of the
Su-2E4 antibody. Murine MAb 2.73 revealed stronger
reactivity with the 68K polypeptide and similar crossreactivity with the 60K polypeptide and another polypeptide in the 120K region. Partially purified (U1)sn
RNP preparations were then used for immunoblotting
to confirm the activity of Su-2E4 against the
(U1)snRNP 68K polypeptide.
Several MCTD reference sera with anti-(U 1)
snRNP activities recognized 68K, as well as A and C
(Figure I , lanes 1-4). One serum also stained the Sm
bands, BIB’ and D (Figure 1, lane 2). Lane 3 in Figure
1 shows the serum pattern of the patient who donated
the lymphocytes for Su-2E4 isolation. The major band
Supernatant Dilution
Figure 2. Enzyme-linked immunosorbent assay using purified (U 1)
small nuclear RNP (snRNP) polypeptides as antigens. Wells precoated with various (U 1)snRNP polypeptides, as shown, were
incubated with dilutions of A , Su-2E4 or B, 2.73 supernatant, and
subsequently reacted with A, anti-human IgM ( p chain-specific) or
B, anti-mouse IgG (heavy and light chain-specific).
Figure 1. Immunoblotting of anti-(UI) small nuclear RNP (snRNP)
antibodies. Affinity-purified (U1)snRNP was used as the antigen for
immunoblotting. Antibodies used were patient sera with known
anti-snRNP reactivities (lanes 1 4 ) , human Su-2E4 (lane 3 , and
murine 2.73 (lane 6 ) . The molecular weight designations on the left
were based on molecular weight standards, and the .polypeptide
.. .
notations on the right were based on reference snRNP sera (16).
recognized by Su-2E4 was the 68K polypeptide
(Figure 1, lane 5 ) , and was similar to the pattern
revealed by murine MAb 2.73 (Figure 1, lane 6), which
was previously reported to react with the (U1)snRNP
68K polypeptide (26,36).
Anti-68K polypeptide ELISA activity. A polypeptide ELISA assay with 68K, A, B/B’, or D polypeptide individually coated onto microtiter plates
showed Su-2E4 to have a high level of binding to the
68K polypeptide. A 32-fold dilution of Su-2E4 supernatant still resulted in significant binding. Polypeptides
A, BIB’, and D were not bound significantly by Su-2E4
antibody (Figure 2A), indicating that the (U1)snRNP
reactivity of Su-2E4 is specific for the 68K polypeptide. When murine anti-(U1)snRNP MAb 2.73 was
similarly tested, its specific binding to 68K was revealed (Figure 2B), and no A, B/B’, or D was bound by
2.73 (data not shown).
Competition analysis of the anti-68K reactivity. Individual (U 1)snRNP polypeptides were used in
ELISAs to compete for Su-2E4 binding to the 68K
polypeptide. Each polypeptide preparation was previously found to inhibit binding between appropriate
anti-(U1)snRNPpatient sera and the same polypeptide
(17) (data not shown). Various concentrations of each
competing polypeptide were first incubated with Su2E4 supernatant. The antibody-antigen
mixture was
then used as the source of primary antibody in poly-
CHEN ET AL
1268
B
A
70
-0
1
-0
48
24
12
6
3
1.5
48
24
12
6
3
Sm MAb Y 12 precipitated the U1, U2, U4, U5, and U6
RNAs (Figure 5, lane S), and the 2.73 MAb precipitated only the U l RNA (Figure 5, lane 3), as previously reported (31,36). Since Su-2E4 is of IgM class,
modifications of conventional techniques for IgG immunoprecipitation were used (see Materials and Methods). Su-2E4 was able to precipitate only the U1 RNA
(Figure 5 , lane 4).
Su-2E4 antibody was also used to stain various
cell preparations by immunofluorescence. At the antibody concentrations at which significant reactivities
could be observed by ELISA, immunoblotting, and
immunoprecipitation, no distinct immunofluorescence
pattern was revealed.
DISCUSSION
Inhibiting 68K Polypeptide (Uglml)
Figure 3. Competitive 68K polypeptide enzyme-linked imrnunosorbent assay results. A, Su-2E4 or B, 2.73 supernatant was first
incubated with various concentrations of 68K polypeptide, and then
applied to wells precoated with the same polypeptide, followed by
incubation with A, anti-human IgM ( p chain-specific) or B, antimouse IgG (heavy and light chain-specific).
peptide ELISA. Preincubation of Su-2E4 antibody
with the 68K polypeptide showed up to SO% inhibition
of Su-2E4 binding to the immobilized 68K (Figure 3A).
Other (U1)snRNP polypeptides tested (A, B/B', and
D) did not affect the ability of Su-2E4 to bind 68K (data
not shown), as would be expected since Su-2E4 antibody failed to bind these polypeptides (Figure 2A).
Similarly, the binding of murine MAb 2.73 to immobilized 68K was inhibited up to 62% by preincubation of
2.73 supernatant with the competing 68K polypeptide
(Figure 3B).
Because both Su-2E4 and 2.73 antibodies reacted specifically with the 68K polypeptide, the epitope
relationship between the 2 antibodies was analyzed.
Microtiter plates coated with the 68K polypeptide
were incubated first with various dilutions of 2.73
supernatant, then with Su-2E4 antibody. The effect of
MAb 2.73 on the binding of Su-2E4 to 68K was
monitored by the use of horseradish peroxidase-conjugated anti-human IgM ( p chain-specific) and subsequent color development. The murine MAb effectively
competed for the binding of Su-2E4 to 68K, with up to
57% inhibition (Figure 4).
RNA immunoprecipitation. Su-2E4 antibody
was further analyzed by RNA immunoprecipitation
along with murine MAbs 2.73 and Y12. Murine anti-
We have described the isolation and characterization of a human autoantibody secreted by a human
lymphocyte cell line immortalized by in vitro EBV
transformation of lymphocytes from a patient with
MCTD. This human autoantibody, Su-2E4, reacts
6o 1
n
50 -
s
W
c
0
:
E 409
.
I
m
c
c
I
-
30 -
20
1 :1
1 :4
1:16
1:64
Dilution of 2.73 Supernatant
Figure 4. Competition of Su-2E4 anti-68K activity by monoclonal
antibody 2.73. Wells precoated with 68K polypeptide were incubated with various dilutions of 2.73 supernatant, followed by undiluted Su-2E4 supernatant, anti-human IgM ( F chain-specific), and
substrate.
ANTI-68K POLYPEPTIDE REACTIVITY
Figure 5. RNA immunoprecipitation with anti-small nuclear RNP
antibodies. Immunoprecipitated 32P-labeled RNAs were electrophoretically separated and exposed on autoradiograms. Antibodies used
were culture medium as negative controls (lanes 1 and 2), murine
2.73 (lane 3), human Su-2E4 (lane 4), and murine Y12 (lane 5).
Anti-IgM intermediate antibody was used for lanes 2 and 4. tRNA =
transfer RNA.
specifically with the (U 1)snRNP-specific 68K polypeptide and precipitates only the U1 RNA. We are not
aware of any previously published reports on human
autoantibodies produced by immortalized human lymphocyte cell lines against this polypeptide. The binding
specificity of Su-2E4 was verified by immunoblotting,
polypeptide ELISA, and RNA immunoprecipitation.
Murine MAb 2.73, known to bind the 68K polypeptide
(26), effectively competed with this human autoantibody.
Imrnunoblotting with Su-2E4 against Jurkat cell
extract revealed only weak staining of 68K (data not
shown); however, intense staining of this polypeptide
was obtained with the use of rabbit (U1)snRNP as the
antigen (Figure 1). This antigen was affinity-purified
1269
using an anti-(U1)snRNP serum from a patient with
MCTD. For immunoprecipitation of U snRNA, it was
necessary to modify conventional techniques for IgG
antibodies, since Su-2E4 antibody is of the IgM class.
ELISA using isolated (U 1)snRNP polypeptides
is a specific and quantitative assay for antibodies
against individual (U1)snRNP polypeptides (17). Each
polypeptide preparation used was shown to contain no
contaminating polypeptides when analyzed by SDSPAGE (17). Both human Su-2E4 and murine 2.73
showed significant and specific reactivity only with
68K in the polypeptide ELISA. This specificity was
confirmed by competitive polypeptide ELISA, where
only 68K competed. Competition between Su-2E4 and
2.73 antibodies was analyzed by preincubation of
68K-coated wells with 2.73 before the addition of
Su-2E4, according to the method of Reuter and Luhrmann (28). Using this method, any difference between
2 antibodies’ affinity for the antigen is likely to have
less influence than when the 2 antibodies are placed
into a well simultaneously.
The binding of Su-2E4 to 68K was significantly
inhibited by preincubation of the antigen with 2.73
(Figure 4). It is therefore possible that Su-2E4 and 2.73
react with a common epitope on the (U1)snRNPspecific 68K polypeptide. Alternatively, the epitopes
recognized by Su-2E4 and 2.73 may be in such close
proximity that simultaneous binding is inhibited for
steric reasons. The incompleteness of the inhibition
suggests that these 2 antibodies may not bind to
identical epitope(s) on the 68K polypeptide.
The snRNP complexes are well known for their
association with some important biologic functions
(8,9). Recent studies have shown strong associations
between autoantibodies to the 68K polypeptide and
MCTD, as well as between autoantibodies to the B/B’
and D polypeptides and SLE (16,17). MAbs to polypeptides in these complexes are very useful in the
investigation of the biochemical and antigenic structures of the complexes. Cloning of complementary
DNA encoding the 68K polypeptide has led to new
information regarding the nucleic acid sequence and
inferred amino acid sequence of this polypeptide
(37,38). In addition, a region of homology has been
found in the 68K polypeptide that is cross-reactive
with retroviral p30gag protein antigen, which could
indicate a role for retroviruses in the initiation of
autoimmunity (37). The availability of both human and
murine autoantibodies secreted by immortalized cell
lines to the 68K polypeptide and other snRNP antigens
CHEN ET AL
1270
will facilitate further studies o n the nature of autoimmune reactivity and its origin in patients with systemic rheumatic diseases.
REFERENCES
1. Tan EM: Autoantibodies to nuclear antigens (ANA):
their immunology and medicine. Adv Immunol 33: 167240, 1982
2. Sharp GC, Alspaugh MA: Autoantibodies to nonhistone
nuclear antigens: their immunobiology and clinical relevance, Immunology of Rheumatic Diseases. Edited by S
Gupta, N Talal. New York, Plenum, 1985
3. Wiik A: The value of specific ANA determination in
rheumatology: update 1986. Allergy 42:241-261, 1987
4. Hardin JA, Mimori T: Autoantibodies to ribonucleoproteins. Clin Rheum Dis 11:485-505, 1985
5. Sharp GC, Irvin WS, Tan EM, Could RG, Holman HR:
Mixed connective tissue disease: an apparently distinct
rheumatic disease syndrome associated with a specific
antibody to an extractable nuclear antigen (ENA). Am J
Med 52: 148-159, 1972
6. Notman DD, Kurata N, Tan EM: Profiles of antinuclear
antibodies in systemic rheumatic diseases. Ann Intern
Med 83:464469, 1975
7. Sharp GC, Irvin WS, May CM, Holman HR, McDuffie
FC, Hess EV, Schmid FR: Association of antibodies to
ribonucleoprotein and Sm antigens with mixed connective tissue disease, systemic lupus erythematosus and
other rheumatic diseases. N Engl J Med 295: 1149-1 154,
1976
8. Maniatis T , Reed R: The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature
325:673-678, 1987
9. Sharp PA: Splicing of messenger RNA precursors. Science 235:76&771, 1987
10. Billings PB, Hoch SO: Characterization of U small
nuclear RNA-associated proteins. J Biol Chem 259:
12850-12856, 1984
11. Mimori T, Hinterberger M, Pettersson I, Steitz JA:
Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap
syndrome. J Biol Chem 259:56&565, 1984
12. Pettersson I, Hinterberger M, Mimori T, Gottlieb E,
Steitz JA: The structure of mammalian small nuclear
ribonucleoproteins: identification of multiple protein
components reactive with anti-(U1)ribonucleoprotein
and anti-Sm autoantibodies. J Biol Chem 2595907-5914,
1984
13. Habets W, Hoet M, Bringmann P, Luhrmann R, van
Venrooij WJ: Autoantibodies to ribonucleoprotein particles containing U2 small nuclear RNA. EMBO J
4: 1545-1550, 1985
14. Lerner MR, Steitz JA: Antibodies to small nuclear
RNAs complexed with proteins are produced by pa-
tients with systemic lupus erythematosus. Proc Natl
Acad Sci USA 765495-5499, 1979
15. Van Venrooij WJ: Autoantibodies against small nuclear
ribonucleoprotein components. J Rheumatol 14 (suppl
13):78-82, 1987
16. Pettersson I, Wang G, Smith EI, Wigzell H, Hedfors E,
Horn J, Sharp GC: The use of immunoblotting and
immunoprecipitation of (U) small nuclear ribonucleoproteins in the analysis of sera of patients with mixed
connective tissue disease and systemic lupus erythematosus: a cross-sectional, longitudinal study. Arthritis
Rheum 29:986-996, 1986
17. Takeda Y, Wang GS, Wang RJ, Anderson SK, Pettersson I, Amaki S, Sharp GC: Enzyme linked immunosorbent assay using isolated (U) small nuclear ribonucleoprotein polypeptides as antigens to investigate the
clinical significance of autoantibodies to these peptides.
Submitted for publication
18. Kohler G, Milstein C: Continuous culture of fused cells
secreting antibody of predefined specificity. Nature
256:495497, 1975
19. Rosen A, Gergely P, Jondal M, Klein G, Britton S:
Polyclonal Ig production after Epstein-Barr virus infection of human lymphocytes in vitro. Nature 267:52-54,
1977
20. Steinitz M, Klein G, Koskimies S, Make1 0: EB virusinduced B lymphocyte cell lines producing specific antibody. Nature 269:420-422, 1977
21. Rosen A, Persson K, Klein G: Human monoclonal
antibodies to a genus-specific chlamydia1 antigen, produced by EBV-transformed B cells. J Immunol 130:
2899-2902, 1983
22. Eilat D: Monoclonal autoantibodies: an approach to
studying autoimmune disease. Mol Immunol 19:943955, 1982
23. Pisetsky DS: Hybridoma SLE autoantibodies: insights
for the pathogenesis of autoimmune disease. Clin Immuno1 Rev 3:169-234, 1984
24. Shoenfeld Y, Schwartz RS: Immunologic and genetic
factors in autoimmune diseases. N Engl J Med 311:
1019-1029, 1984
25. Chiorazzi N: Human monoclonal antibodies as probes to
study autoimmune and allergic disorders. Bio/Technology 4:210-218, 1986
26. Billings PB, Allen RW, Jensen FC, Hoch SO: Anti-RNP
monoclonal antibodies derived from a mouse strain with
lupus-like autoimmunity. J Immunol 128:1176-1 180,
1982
27. Pisetsky DS, Salistad DM, Chambers JC: Characterization and idiotypic analysis of an anti-RNP monoclonal
antibody. Clin Exp Immunol 56593-600, 1984
28. Reuter R, Luhrmann R: Immunization of mice with
purified U 1 small nuclear ribonucleoprotein (RNP) induces a pattern of antibody specificities characteristic of
the anti-Sm and anti-RNP autoimmune response of
ANTI-68K POLYPEPTIDE REACTIVITY
29.
30.
31.
32.
33.
patients with lupus erythematosus, as measured by
monoclonal antibodies. Proc Natl Acad Sci USA 83:
8689-8693, 1986
Reuter R, Lehner CF, Nigg EA, Luhrmann R: A monoclonal antibody specific for snRNPs U1 and U2. FEBS
Lett 201:25-30, 1986
Wise KS, Watson RK: Mycoplasma hyorhinis GDL
surface protein antigen p 120 defined by monoclonal
antibody. Infect Immun 41 :1332-1339, 1983
Lerner ER, Lerner MR, Janeway CA Jr, Steitz JA:
Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc Natl Acad Sci USA 78:27372741, 1981
Billings PB, Hoch SO: Isolation of intact SmiRNP
antigens from rabbit thymus. J Immunol 131:347-35 1,
1983
Laemmli UK: Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:
680-685, 1970
1271
34. Treadwell EL, Boak AM, Kovacs SAH, Chen J, Wang
RJ,Sharp GC, Agris PF: The autoimmune antigen Me is
distinct and related to undifferentiated connective tissue
disease. Arthritis Rheum 30: 123S1246, 1987
35. Hinterberger M, Pettersson I, Steitz JA: Isolation of
small nuclear ribonucleoproteins containing U 1, U2,
U4, U5, and U6 RNAs. J Biol Chem 258:2604-2613,
1983
36. Winkler A, Watson-McKown R, Wise KS: Monoclonal
autoantibody recognizing a unique set of small nuclear
ribonucleoprotein complexes. J Immunol 140:69-77,
1988
37. Query CC, Keene JD: A human autoimmune protein
associated with U l RNA contains a region of homology
that is cross-reactive with retroviral p30gagantigen. Cell
51:211-220, 1987
38. Theissen H, Etzerodt M, Reuter R, Schneider C, Lottspeich F, Argos P, Luhrmann R, Philipson L: Cloning of
the human cDNA for the U l RNA-associated 70K
protein. EMBO J 5:3209-3217, 1986
Документ
Категория
Без категории
Просмотров
0
Размер файла
600 Кб
Теги
immortalized, 68k, nuclear, ribonucleoproteins, secreted, small, polypeptide, autoantibody, human, lymphocytes, line, cells
1/--страниц
Пожаловаться на содержимое документа