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Detection of antibodies to small nuclear ribonucleoproteins and small cytoplasmic ribonucleoproteins using unlabeled cell extracts.

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1356
DETECTION OF ANTIBODIES TO
SMALL NUCLEAR RIBONUCLEOPROTEINS AND
SMALL CYTOPLASMIC RIBONUCLEOPROTEINS
USING UNLABELED CELL EXTRACTS
MARK S. FORMAN, MARY NAKAMURA, TSUNEYO MIMORI, CARMEN GELPI,
and JOHN A. HARDIN
RNA molecules immunoprecipitated with sera
from patients who have rheumatic diseases can be
readily detected in polyacrylamide gels by using
ethidium bromide and silver stains. With these stains,
we found that RNA patterns characteristic of a broad
range of specific small nuclear ribonucleoproteins and
small cytoplasmic ribonucleoproteins were recognizable. The stains correctly identified antibodies to ribonucleoproteinsin 33 (92%)of 36 patient sera selected for
study because of known antibody specificities. The silver
stain method detected antibodies to ribonucleoproteins
in 25 (76%) of 33 patients with classic systemic lupus
erythematosus, a frequency that approximated the frequency observed in the Lerner-Steitz assay, which is
based on autoradiography. This approach considerably
simplifies the latter radioimmunoassay with minimal
loss of precision and sensitivity.
Patients who have rheumatic diseases produce
a broad range of autoantibodies that bind specific
protein components of different ribonucleoprotein
(RNP) particles (1). Detection of these antibodies can
often be helpful clinically. However conventional tests
based on immunodiffusion, counterimmunoelectrophoresis, or hemagglutination are limited by the lack
of biochemically pure nucleoprotein antigens and the
large array of antibodies. Recently, Lerner and Steitz
described a new assay that used test serum to immunoprecipitate specific small nuclear RNP (snRNP)
and small cytoplasmic RNP (scRNP) from extracts of
32P-labeledtissue culture cells (2). This assay provides
an assessment of the total array of antibodies to
snRNP and scRNP in a patient’s serum in a single test
(3). However, the requirement of radiolabeled cells
has largely limited its use to that of a research tool. In
the present paper, we describe an adaptation of the
test that retains much of the sensitivity and specificity
of the radioimmunoassay, yet avoids the use of
radioisotopes.
~
From the Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut.
Supported by grants from the U.S. Public Health Service
(AM-32549, AM-10493, and AM-07107), the Kroc Foundation, the
Arthritis Foundation, the Connecticut Chapter of the Lupus Foundation, and the US.-Spain Joint Committee, and by a donation
from the Permut family.
Mark S . Forman: Student, Yale University; Mary
Nakamura: Student, Yale University School of Medicine; Tsuneyo
Mimori, MD, PhD: Postdoctoral Fellow, Section of Rheumatology ;
Carmen Gelpi, PhD: Visiting Scientist (permanent address: Department of Immunology, Hospital de la Santa Creu i Sant Pau, Padre
Claret, Barcelona, Spain); John A. Hardin, MD: Associate Professor of Medicine, and recipient of an Arthritis Senior Investigator
Award from the Arthritis Foundation.
Address reprint requests to John A. Hardin, MD, Yale
University School of Medicine, 333 Cedar St. (609 LCI), New
Haven, CT 06510.
Submitted for publication September 24, 1984; accepted in
revised form May 28, 1985.
Arthritis and Rheumatism, Vol. 28, No. 12 (December 1985)
MATERIALS AND METHODS
Sera. Serum samples were collected from patients
with systemic lupus erythematosus (SLE) and other rheumatic diseases and were stored at -70°C until used. Standard antibody specificities have been described previously
(3). In selecting patients with SLE, the revised criteria of the
American Rheumatism Association (ARA) were applied (4).
Cell extracts. HeLa cells were grown to a concentration of 2-3 x lo5 cells/ml at 37°C in RPMI 1640 culture
medium (containing 100 pghl of streptomycin, 60 pg/ml of
penicillin, and 5% heat-inactivated newborn calf serum) in
5% C02. The cells were harvested and washed in Tris
buffered saline (150 mM NaCI, 10 mM Tris-HCI, pH 7.4)
using centrifugation at 400g for 3 minutes. They were
suspended in NET-2 buffer (50 mM Tris-HC1, 150 mM NaC1,
0.05% NP-40, pH 7.4) at a concentration of 2 x lo7 cells/ml
DETECTION OF snRNP AND scRNP ANTIBODIES
and sonicated in an ice bath 3 times, for 40 seconds each,
with a Branson sonifier on setting 3. The sonicate was
centrifuged at 12,000g for 30 minutes at 4°C to remove
debris.
Immunoprecipitation. Ten microliters of test sera
was mixed with 2 mg of staphylococcalprotein A-Sepharose
CL-4B (Pharmacia, Piscataway, NJ) in 500 p1 of immunoprecipitation (IPP) buffer (10 mM Tris-HC1, 500 mM
NaCl, 0.1% NP-40, pH 8.0) and rotated (Labquake shaker;
Lab Industries, Berkeley, CA) for 2 hours at 4°C. The
Sepharose particles, which now carried adsorbed IgG, were
washed 3 times in 500 pI of IPP buffer using 10-second spins
in an Eppendorf microfuge, then resuspended in 200 pl of
NET-2 buffer. This suspension was combined with 100-300
pl of HeLa cell extract and again incubated on the rotator for
2 hours at 4°C. The Sepharose particles were then collected
with a 10-second centrifugation in the microfuge, and
washed 3 times with NET-2 buffer as before.
To extract bound nucleic acids, the washed Sepharose particles were resuspended in 300 pl of NET-2
buffer, to which was added 30 pl of 3M sodium acetate, 15 pl
of 20% sodium dodecyl sulfate, and 300 pl of phenol/chloroforrrdisoamyl alcohol (50: 50: 1; containing 0.1% hydroxyquinoline). The tubes were agitated on a vortex and spun for
1 minute in the microfuge. The aqueous phase was recovered
and combined with 900 pl of cold 100% ethanol for 1 hour at
-70°C in dry ice. The tubes were spun in the microfuge for
5 minutes at room temperature, the ethanol was aspirated,
and the pellet (faintly visible) washed once with cold 95%
ethanol. The pellets were vacuum-dried and dissolved in 20
pl of electrophoresis sample buffer which consisted of 1OM
urea, 0.025% bromphenol blue, and 0.025% xylenecyanol FF
in Tiis-borate-EDTA buffer (TBE; 90 mM Tris-HC1, 90 mM
borate, 1 mM EDTA, pH 8.6). The samples were denatured
at 65°C for 3 minutes, then resolved in 7 M urea-10%
polyacrylamide gels in TBE that were 0.7-mm X 16-cm X
12-cim (thickness, width, length). Ten microliters of each
sample was loaded per lane. Electrophoresis was carried out
at 4C0 volts until the second dye front reached the bottom of
the gel. To prevent RNase contamination, all buffers and
plasfic utensils were autoclaved and all glass utensils were
silanized and baked at 200°C for 2 hours.
RNA stains. Following electrophoresis, the gels were
incubated with ethidium bromide (0.5 pg/ml in H20) for 5
minutes and destained in double-distilled water for 5 minutes Stained RNA bands were observed by exposing the
gels to high-intensity ultraviolet light at 300 nm. The results
were photographed with a Polaroid M4 camera.
For staining with silver, the gels were washed in
glass-distilledwater for 1-2 hours, incubated for 5 minutes in
oxidizing reagent (0.0034M potassium dichromate, 0.0032M
nitric acid in water), washed 4 times for 30 seconds in
glass-distilled water until clear, and immersed in a solution
of silver nitrate (0.012M) in water for 30 minutes. During the
first 5 minutes of exposure to the latter solution, glass trays
containing the gels were placed on an x-ray view box to
provide uniform exposure to fluorescent light. The gels were
rinsed twice in water, then once in developer (0.28M sodium
carbonate, 0.5 mllliter of formalin in water) and agitated in
developer until the desired stain intensity was achieved. The
stained gels were then fixed in 1% acetic acid.
1357
Other procedures. Sera were tested in double immunodiffusion using calf thymus extracts as antigens (5) and
by the Lerner-Steitz assay described previously (2).
RESULTS
Both ethidium bromide and silver stains readily
detect RNA molecules that are present in sufficient
quantity (6,7). Initially, we sought to determine if
these stains could detect diagnostic patterns of RNA
molecules in gels containing RNAs extracted from
immunoprecipitates prepared with anti-U 1 RNP and
anti-Sm sera. Optimum results were obtained when
300 pl of cell extract (prepared from 6 x lo6cells) were
precipitated with 2 mg of protein A-Sepharose previously coated with IgG from 10 pl of patient sera. This
method was adopted for use throughout the remainder
of this study. Compared with the earlier radioimmunoassay (2), it represents an equivalent amount of
antibody and a threefold increase in the amount of cell
extract, deletion of carrier RNA during the step in
which phenol-extracted RNAs are precipitated with
ethanol, and use of a shorter gel for resolution of
precipitated RNAs.
Figures 1 and 2 show examples of different
types of immunoprecipitates that have been fractionated electrophoretically and stained. It can be seen
that anti-ribosomal, anti-Sm, anti-U 1 RNP, and
anti-transfer RNA (tRNA) antibodies (which bind the
most abundant RNPs in total cell extract), as well as
anti-La and anti-Ro antibodies (whose antigenic RNPs
contain RNAs that are not even visible in the total cell
extract) precipitated RNAs in sufficient quantities so
that characteristic patterns could be observed. The
silver stain more readily demonstrated the minor RNA
species in the total cell extract and in the highly
banded L a immunoprecipitates, and thus appeared to
provide a somewhat more sensitive detection system.
Previous studies have shown that Ro RNAs compose
a subset of L a RNAs (8). In stained gels, these
patterns could be readily differentiated because La
precipitates contained a large number of minor RNA
species and 2 major RNAs (one migrating just ahead of
the Y4 RNA and the other migrating just behind 5s
ribosomal RNA) that were not present in Ro precipitates.
Using both stains, we were able to detect
additional antibodies that precipitated at least 2 other
subsets of tRNAs, the U3 RNA, and the Th RNA (2)
(data not shown). These latter 2 RNAs are components of nucleoli (9,lO). Thus, nearly all of the small
RNA species that can be detected in immunoprecipi-
1358
Figure 1. Gel stained with ethidium bromide. The major RNA
species present in total cell extract are visible at the left. Normal
human serum (NHS) precipitates no RNAs. Patient sera containing
autoantibodies with the specificities listed in lanes 2-7 precipitate
characteristic subsets of RNAs. 2 = anti; tRNA = transfer RNA.
tates by autoradiography can also be detected by these
simple staining methods.
Table 1 compares the sensitivity of the assay
described here with that of conventional immunodiffusion. Four sera which contained either anti-U1
RNP, anti-Sm (plus anti-U1 RNP activity), anti-Ro, or
anti-La (plus some anti-Ro activity) antibodies were
diluted serially to determine the maximal dilution at
which a positive test result could be obtained. With the
ethidium bromide stain, complete diagnostic RNA
patterns could be recognized at serum dilutions which
equalled (in I case) or exceeded (in 3 cases) those
dilutions that gave positive results with immunodiffusion. The silver stain demonstrated these RNAs at
even higher serum dilutions. In immunoprecipitates
made with anti-Sm antibodies, the U1 RNA was
detectable at much higher titers than were the other
Sm RNAs (U2, U4, U5, and U6). Perhaps this observation reflects the combined presence of anti-U I RNP
FORMAN ET AL
Figure 2. Gel stained with silver stain. The sera are the same as
those used in Figure I . See Figure 1 for definitions.
and anti-Sm antibodies in such sera, as well as the
relative abundance of the U1 RNP particle in cell
extracts.
It is clear from the data in Table 1 that the
Lerner-Steitz assay detects antibodies to RNPs at
serum dilutions that exceed those that give positive
results with the stains. In order to assess the diagnostic significance of this difference in sensitivity, we
examined sera from 36 randomly chosen patients with
various rheumatic diseases. These sera possessed different types of anti-snRNP and anti-scRNP antibodies,
Table 1. Comparison of sensitivity of imrnunoprecipitation assays
and immunodiffusion*
~
~
~
~
Immune- Ethidium
Specificity
diffusion
bromide
Silver
12P
U I RNP
U 1 RNPiSm
I:l
I : 16/l: I
1 :64
I : 1611:4
1:16
I :64/l: 16
1:64
I :64/1:4
1: I ,024
I :1,02411:64
1:256
1: 1,024
I :1,024iI :1,024
I :1.024
1:64/1:4
1:1,024/1:1,024
Ro
Ro/La
* Values represent
maximal dilution that gives a positive result.
DETECTION OF snRNP AND scRNP ANTIBODIES
Table 2. Comparison of staining techniques and autoradiography
in screening of antinuclear antibodies
No’
of
sera
tested
Specilicity in 32P
screen*
Anti-SdU 1 RNP
Anti-U1 RNP
Anti-L,aiRo
Anti-R.o
Anti-tRNA
Anti-ribosome
Anti-U3
Anti-Th
~~
No. positive using other tests
Ethidium
bromide
Silver
stain
Immunodiffusion
4
7
3
10
6
1
1
1
4
7
3
10
6
1
1
1
4
2
3
8
1359
antibodies, and 1 of 11 anti-Ro antibodies. Immunodiffusion was much less sensitive, being positive in only
18 (55%) of the patients. It did not detect 5 of 15
anti-U1 RNP antibodies, 6 of 11 anti-Ro antibodies,
both of the antibodies to ribosomes, or the antibodies
that precipitated the Th RNA and the U3 RNA.
DISCUSSION
5
1
0
0
~~
*tRNA = transfer RNA.
determined by the Lerner-Steitz assay (Table 2). Only
3 sera-1 of 8 anti-U1 RNP, 1 of 11 anti-Ro, and 1 of
7 anti-tRNA-failed to precipitate detectable quantities of RNAs. All 3 of these sera had given only weakly
positlive results in the assay using radiolabeled cell
extracts. Although silver appears to stain more sensitively than ethidium bromide, both stains were equally
effective in identifying antibody specificities among
the sera studied here. In contrast, immunodiffusion
was often deficient, especially in the ability to detect
antibodies to U1 RNP.
It was thought that the performance of the
assays described above might have been biased by a
tendency to select more strongly reactive sera for
study. Consequently, a representative group of patients with SLE were examined. In compiling the
group to be studied, one of us (MN), who had no
knowledge of the previous antibody results, reviewed
the clinical records of all patients; this was followed by
a second review by another author (JAH). All individuals .who unequivocally fulfilled the ARA criteria for
SLE were identified. Patients who were initially referred because of a serologic abnormality and those
with (clinicalfeatures of Sjogren’s syndrome, myositis,
or scleroderma were excluded from the study. Of the
remaining 33 patients (15 of whom were members of
the group depicted in Table 2), 32P-immunoprecipitation assay detected antibodies to 1 or more different
ribonucleoproteins in 26 (79%). In comparison, the
silver stain assay was positive in 25 (76%). However,
among all of the antibodies detected in the 32Passay,
silver staining failed to detect 1 of 3 anti-La antibodies,
the 1J3 antibody (the anti-U3 antibody detected in
Table: 2 was from a different patient with scleroderma),
1 of 2! anti-ribosomal antibodies, 1 of 15 anti-U1 RNP
The Lerner-Steitz assay has provided a highly
sensitive and very specific method for characterizing
antigen-antibody systems that involve ribonucleoprotein particles. For example, antibodies that specifically
precipitate U1, Sm, Ro, and La RNAs, as well as
ribosomal RNA, 5s ribosomal RNA, Th RNA, U3
RNA, U2 RNA, and several subsets of tRNAs, can be
readily identified in patient sera, using this method
(11,12). Approximately 80% of our SLE patients had
antibodies to different ribonucleoproteins that were
detectable in this assay. Moreover, this assay has been
Table 3. Ribonucleoproteins recognized by sera from 33 systemic
lupus erythematosus patients, using different assays
Patient
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27-33
*
t
32p
Silver stain
UI, Sm, Ro*
Ul,* Sm,Ro*
UI, Sm,Ro,* Lat
UI, Sm,Ro*
UI, Sm
U1, Sm
UI*, Sm
UI, U3t
u1
UI
u1*
Ul*
UI*
UI, Ro*
Ult, Ro*
Ro, Th*
Rot
Ro
Ro*
Ro
Ro*
Ro*
Ro*
La
La
rRNA*§
-
UI,
UI,
UI,
UI,
Sm,Ro
Sm, Ro
Sm, Ro
Sm, Ro
UI, Sm
UI, Sm
UI, Sm
UI
UI
u1
UI
UI
UI
UI, Ro
Ro
Ro, Th
Ro
Ro
Ro
Ro
Ro
Ro
La
La
rRNA
-
Immunodiffusion
UI, Sm
Sm
UI, Sm
UI, Sm
UI, Srn
UI, Sm
Sm
UI
UI
u1
u1
u1
-
Ro
Ro
Ro
Ro
-
-
La
La
-
not detected by immunodiffusion assay.
not detected by silver stain assay, nor by immunodiffusion
assay.
t = not detected by silver stain assay.
§ rRNA = ribosomal RNA.
=
=
1360
a powerful tool for characterizing the physical nature
of these antigens (13). Thus, the assay is capable of
providing the clearest picture possible of the total
spectrum of these antibodies in patient serum.
The present study shows that simple staining
procedures can be used to visualize immunoprecipitated RNAs, with little loss of clinically significant sensitivity and retention of ability to discriminate among a
broad spectrum of different autoantibodies. Similar
observations have recently been reported for anti-U 1
R N P and anti-La antibodies (14). The assay is clearly
far more sensitive and specific than immunodiffusion.
However, it can be noted from the data given in Table
3, from a relatively small group of patients, that
immunodiffusion appears to be a very satisfactory
method for detecting anti-Sm antibodies.
The assay described here can be performed in a
single day. However, it can be conveniently carried
out over 2 days; the ethanol-precipitated RNAs can be
stored overnight at -20°C and the gel electrophoresis
and the staining procedures performed the following
morning. Since extracts of radiolabeled cells undergo
spontaneous degradation within a few days, we have
always used freshly prepared substrate. However with
unlabeled cell extracts, possibly in combination with
RNase inhibitors, it might be possible to store batches
of substrate for use in repeated assays.
The major limitations of this assay are the same
as those of the earlier assay that was based on
autoradiography. Only semiquantitative results are
obtained unless test sera are assayed with serial dilutions. Also, because the U 1 R N P epitopes are located
on the same R N P particle a s the Sm epitopes, it can be
difficult to detect anti-U1 R N P in the presence of
anti-Sm antibodies since both antibodies precipitate
U1 RNA (15). However, this may not be a serious
clinical disadvantage since a modification of the
radioimmunoassay (15), as well as earlier data (16),
demonstrate that anti-U 1 R N P antibodies occur in
nearly all patients with anti-Sm antibodies. The same
situation applies to the Ro-La system, where a subset
of La RNAs carry both the Ro and La antigens (17).
These 2 antibodies also occur in linked sets since
anti-Ro antibodies have been reported to occur in
nearly all patients with anti-La antibodies (18). Thus,
the staining methods described here provide a convenient method of analysis of antibodies to ribonucleoproteins, which can be performed in any clinical
laboratory.
FORMAN ET AL
ACKNOWLEDGMENTS
We thank Dr. Stephen E. Malawista and Professor
Joan A. Steitz for helpful advice and discussion during the
course of this work, and Mary Ann Livieri for preparation of
the manuscript.
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DETECTION OF snRNP AND scRNP ANTIBODIES
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1361
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