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The detection and identification of antibodies to saline extractable nuclear antigens by counterimmunoelectrophoresis.

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1026
THE DETECTION AND IDENTIFICATION
OF ANTIBODIES TO
SALINE EXTRACTABLE NUCLEAR ANTIGENS
BY COUNTERIMMUNOELECTROPHORESIS
HAROLD D. KEISER and JOSHUA WEINSTEIN
Sera containing antibodies to the saline extractable nuclear antigens RNP, Sm, and Ha can be identified with counterimmunoelectrophoresis (CIE) by
matching their precipitin reactions to those of reference
sera. The CIE reaction of Sm is unusual in that two precipitin lines are formed, one of which shows partial immunologic identity with the reaction to RNP. Sera were
identified by CIE which reacted with additional acidic
antigens in rabbit thymus extract, some of which had
the same enzyme sensitivity profiles as RNP and Ha but
were immunologically not identical and could not be detected by hemagglutination with red cells coated with
calf thymus nuclear extract. Although the incidence of
reactivity to RNP, Sm, and Ha was similar to previous
reports, review of the clinical manifestations of 128 patients with systemic lupus erythematosus (SLE) did not
provide statistically significant confirmation of previously reported correlations between the presence and
specificity of reactivity and particular patterns of disease expression. With due attention paid to technical
artifacts, CIE is a sensitive and specific method for identifying sera reactive with acidic nuclear antigens that
has practical advantages over alternative methods.
From the Department of Medicine, Albert Einstein College
of Medicine, Bronx, New York.
Supported by research awards from the S.L.E. Foundation of
America and Research Career Development Award AM00024 of the
National Institutes of Health (H.D.K.).
Harold D. Keiser, MD: Associate Professor of Medicine;
Joshua Weinstein, MD: Assistant Professor of Medicine, Albert Einstein College of Medicine.
Address reprint requests to Harold D. Keiser, MD, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris
Park Avenue, Bronx, New York 10461.
Submitted for publication September 21, 1979; accepted in
revised form February 19, 1980.
Arthritis and Rheumatism, Vol. 23, No. 9 (September 1980)
Many of the antinuclear antibodies in the sera of
patients with connective tissue diseases react with components of mammalian cell nuclei which are solubilized
by normal saline solutions. These antibodies are of considerable interest because they comprise as much as 20%
of total serum immunoglobulin in some patients (l),
and because antibodies to specific saline-extractable nuclear antigens (ENA) have been associated with disease
subgroups with characteristic clinical features and prognoses (2-4). Thus, the presence of antibodies to the nuclear antigen RNP is said to be the hallmark of a group
of patients with overlapping features of systemic lupus
erythematosus (SLE) and another rheumatic disease
who have a low incidence of renal abnormality and a
good prognosis (5,6). Antibodies to the nuclear antigen
Sm seem to be specific for patients with SLE (3,4,7), and
antibodies to the nuclear antigen Ha-also termed SS-B
or La ( 8 b a r e reported to occur with high frequency in
patients with idiopathic or SLE-associated Sjogren’s
syndrome who may be at greater risk of disease progression or of developing pseudolymphoma (9).
The methods most commonly employed in the
detection and identification of antibodies to ENA, agarose double immunodiffusion and hemagglutination,
are not entirely satisfactory. Immunodiffusion is a relatively insensitive technique, and it is usually necessary
to test multiple serum dilutions to be certain of including the proper ratio for antigen-antibody precipitation.
Hemagglutination is a highly sensitive technique, but
Ha, and possibly other ENA, cannot be detected by
hemagglutination (lo), and it may not be possible to
distinguish among the multiple ribonuclease-sensitive
or insensitive ENA which could be responsible for a
hemagglutination reaction.
The studies reported here indicate that counter-
ANTIBODIES TO SALINE ENA
immunoelectrophoresis (CIE), as described by Kurata
and Tan (1 l), may be a more practical method for detecting antibodies to ENA. With proper placement of
wells and recognition of anomolous or nonimmunologic
reactions, sera containing antibodies to RNP, Sm, Ha,
and other ENA, singly or in combination, can be readily and accurately identified by CIE.
MATERIALS AND METHODS
Sera. The serum samples examined for antibodies to
ENA were originally submitted to this laboratory for serum
hemolytic complement and anti-native DNA antibody level
determinations. Only sera of the patients known to have antinuclear antibodies were tested for antibodies to ENA, and
when multiple samples were available from a patient, the
sample examined was chosen from a period of active disease
and/or depressed serum complement. Sera were stored at
-20°C and routinely heated at 56OC for 1 hour prior to testing, although the testing of a dozen serum samples in parallel
suggested that heat inactivation may not be necessary. Reference sera for RNP, Sm, and Ha were provided by Dr. Halstead R. Holman, and a reference serum for Ro was provided
by Dr. Peter Maddison.
Counterimmunoelectrophoresis. CIE was carried out
essentially as described by Kurata and Tan (ll), the major
modification being the arrangement of the wells. The gel cutter consisted of two parallel rows of punches 4 mm in outside
diameter separated by 5 mm from outer edge to outer edge,
with the punches in each vertical row approximately 0.5 mm
apart. The row of wells on the left (cathodal) side was filled
with antigen solution and the row on the right side (anodal)
with sera. Electrophoresis was performed in a Durrum type
apparatus in 0.05M barbital buffer, pH 8.6, with a constant
current of 4 mamp per slide and was stopped when the orange
G marker dye had migrated approximately 15 mm. This took
1 to 1% hours, which is somewhat longer than reported by
Kurata and Tan (11). Slides were read after overnight incubation in a moist chamber at room temperature. Washing
the slides for several days with cold 5% sodium citrate or normal saline occasionally improved the definition of precipitin
1027
lines, but subsequent drying and staining with Coomassie blue
were not helpful.
Hemagglutination. Passive hemagglutination with
sheep red cells sensitized with saline extract of calf thymus
nuclei was performed by Dr. Peter Barland (7).
Nuclear antigens. A commercial preparation of
lyophilized rabbit thymus extract (Pel-Freeze Biologicals,
Rogers, Arkansas) was used as the source of saline-extractable
nuclear antigens. Extraction and enzyme treatment were performed as described by Kurata and Tan (1 1); digestion with
trypsin (DCC-treated, type XI, Sigma Chemical Co., St.
Louis, Missouri), not described by these authors, was performed at a 1 :25 ratio of enzyme to substrate with incubation
in a 1 : 1 volume mixture for 30 minutes at 37°C. Extracts and
digests were stored in aliquots at -20°C for as long as 4
months with no apparent loss of activity. A 0.35M NaCl extract of purified rat liver chmmatin was provided by Dr. Irving Listowsky.
Clinical data. A detailed review was performed of the
clinical features of the 106 of 149 patients with ENA-positive
sera and of the 41 SLE patients with ENA-negative sera
whose medical records were available for analysis. The diagnosis of SLE was established on the basis of the presence of
four provisional American Rheumatism Association criteria
(12) or by the presence of three criteria and one of the following: 1) a positive LE band test (i.e. dermal-epidermal deposits
of immunoglobulins and complement components in clinically uninvolved skin); 2) elevated levels of antibodies to native DNA by the Millipore filter assay (13) with Iz5I-labeled
filtered DNA (14); or 3) classic electron microscopic and immunofluorescence findings on renal biopsy. The diagnoses
of dermato-polymyositis and scleroderma were made on the
basis of the presence of the usual clinical features (15). Patients who met the diagnostic criteria for SLE and in addition
had biopsy, electromyographic, or enzyme evidence of myositis or who had clinical, biopsy, or radiographic evidence of
scleroderma were considered to have overlapping SLE-polymyositis or SLE-scleroderma respectively.
Statistics. The statistical significance of the differences
in incidence of selected clinical parameters between specific
serologically defined groups and all other SLE patients studied was determined by the 2 test.
Figure 1. Counterimmunoelectrophoresiswith rabbit thymus extract (TE) and ribonuclease (RNAse), deoxyribonuclease
(DNAse), and trypsin (Try) digests of thymus extract in the sets of wells on the left (cathodal) and standard reference sera
for RNP (a), Sm (b), and Ha (c) in the sets of wells on the right (anodal).
KEISER AND WEINSTEIN
1028
b o
lo
TE
TE
0
1
0
!O
0
13
0
U
Figure 2. Counterimmunoelectrophoresiswith thymus extract or its digests in the anodal sets of wells as denoted in Figure 1 and patient
sera in the cathodal wells. Sera are denoted by their specificity as determined by enzymatic sensitivity and immunologic matching with
reference sera, with the individual patient’s initials as subscript (e.g., RNPG, denotes serum with RNP reactivity obtained from patient
GP).
RESULTS
RNP, Ha, Sm. Sera known to contain antinuclear antibodies were reacted by CIE with a saline
extract of rabbit thymus and with extract treated with
ribonuclease, deoxyribonuclease, or trypsin. The CIE
results using standard sera for RNP, Sm, and Ha obtained from Dr. H. R. Holman are shown in Figure 1.
Antibodies to RNP form a single sharp precipitin line
which is weak or absent after digestion of the thymus
extract with ribonuclease or trypsin. Antibodies to Ha
also form a single precipitin line, but this reaction is not
affected by treatment of the thymus extract with any of
the enzymes used. By contrast, antibodies to Sm usually
produce both a sharp precipitin line and, adjacent to it
on the side nearer to the antigen well, a broad fuzzy
band or line. The sharper precipitin line of Sm is weak
or absent with ribonuclease-treated thymus extract, but
it persists and even appears enhanced after trypsin digestion of the thymus extract.
Sera reactive with RNP, Ha, or Sm can be definitively identified by CIE on the basis of the relationship
between their precipitin bands and those of standard
sera run in adjacent sets of wells. Thus, it can be appreciated that serum in the middle well in Figure 2A con-
tains antibodies to both RNP and Ha, inasmuch as the
precipitin line nearer to the antigen well merges
smoothly with the line formed by a standard anti-RNP
serum, and the precipitin line nearer to the serum well
merges smoothly with the line formed by a standard
anti-Ha serum. Crossing-over or wide divergence of
precipitin lines, as with those formed by the reactions to
Ha and RNP or Sm in adjacent sets of wells (Figure 2B,
top and bottom), are indicative of immunologic nonidentity. The immunologic relationship of RNP and Sm
as shown by CIE is complex (Figure 2B, middle). Both
of the precipitin lines of Sm cross over the precipitin
line of RNP, but the RNP precipitin line crosses over
only the less distinct Sm precipitin line nearer to the antigen well and appears to merge with the sharper Sm
precipitin line. The resultant spurring of the sharper Sm
precipitin line over the RNP precipitin line indicates
partial immunologic identity. Sera that contain antibodies to both Sm and RNP produce two precipitin
lines upon reaction with thymus extract (Figure 2C). A
standardized RNP precipitin line formed by the reaction in an adjacent set of wells merges with the precipitin line nearer to the antigen well (Figure 2D); both precipitin lines appear to merge with the sharper precipitin
ANTIBODIES TO SALINE ENA
1029
Figure 3. Countetimmunoelectrophoresis with thymus extract or its digests in the anodal sets of wells and patient sera in the
cathodal wells. Notations are the same as in Figures 1 and 2.
line formed by the reaction to Sm in an adjacent set of
wells (Figure 2D).
Additional acidic nuclear antigens. Over the
course of these studies, two or more sera were identified
that reacted by CIE with four additional components of
the thymus extract: 1) RNP-2, a ribonuclease and trypsin sensitive antigen which is immunologically distinct
from RNP (Figures 3A and B); 2) P-2, which is resistant
to digestion with ribonuclease, deoxyribonuclease, or
trypsin but is immunologically distinct from Ha (Figure
3C and D); 3) “DNA,” a reaction sensitive to digestion
with deoxyribonuclease and detected thus far only in
sera with high titers of antibodies to native DNA (Figure 3E); and 4) P-3, which, unlike the other antigens described, is sensitive to trypsin but resistant to ribonuclease (Figure 3F). Several additional antigens have
been detected in the thymus extract which have thus far
been found to react with only a single serum.
The incidence of reactivity to the rabbit thymus
extract and to each of its recognized antigens, as determined by CIE, is shown in Table 1. Of interest is the
frequency with which antibodies to two ENA were
identified by CIE in the same sera (Table 1, bottom).
Thirty percent of sera with anti-RNP and 49 percent of
sera with anti-Sm also reacted with another ENA, the
most common combination being reactivity to both
RNP and Sm.
With the exception of the sera reactive with
DNA and P-3, which were not tested, each of the antigens found by CIE with rabbit thymus extract were also
found with a 0.35M NaCl extract of purified rat liver
chromatin. These antigens were found in the precipitate
formed by the thymus extract in 2% TCA, indicating
that they are components of the poorly characterized
LMG fraction of chromatin (16). A serum reactive with
Table 1. Incidence of ENA reactivities in ANA positive sera
ENA
reactivity
RNP*
smt
Ha
RNP-2
“DNA
P-2
P-3
Unmatched
Number
reactive
sera
87
37
22
10
6
4
2
12
9’0 ENA
positive
sera (total
= 149)
58
25
15
7
4
3
1
8
% ANA
positive
sera (total
= 357)
24
10
6
3
2
1
<I
3
* RNP + another ENA:26 (Sm, 12; Ha, 6; RNP-2, 6; unmatched,
2).
t Sm + another ENA:20 (RNP, 12; Ha, 2; RNP-2, 2; P-2, 1; unmatched, 3).
KEISER AND WEINSTEIN
1030
Figure 4. Counterimmunoelectrophoresiswith empty anodal wells (a) or with thymus extract and its digests in the anodal wells @-d) and patient
sera in the cathodal wells. Sera non-reactive with thymus extract are denoted by ENA-,with individual sera identified by the patient’s initials in the
subscript. The well labeled Hacs-SP (b, bottom) was deliberately overfilled to allow the serum to spill over the well edge. Other notations are the
same as in previous figures.
the cytoplasmic antigen Rc-equivalent to SS-A (8)gave no CIE reaction with the rabbit thymus extract.
Problems of CIE interpretation. In interpreting
the results of CIE, care must be taken to avoid certain
common artifacts. Arcs surrounding the serum well on
the side nearer the antigen are frequently noted and do
not represent an immunologic reaction inasmuch as
they can be found after electrophoresisin the absence of
thymus extract (Figure 4A). Overfilling of a serum well
with consequent spillage into the wells above or below
can produce a spurious precipitin line near those wells,
as shown in Figure 4B. A large, ill-defined precipitin reaction between serum and trypsin-digested thymus extract may occur even with sera that do not react with the
untreated thymus extract, especially after prolonged incubation or washing of the CIE plate (Figure 4C). Finally, the finding of an indistinct precipitin band, delayed in its appearance relative to most of the other
precipitin lines, is not a distinguishing characteristic of
Sm, as has been maintained previously (1 1). Similar
blurred and delayed reactions occur with other thymus
antigens and individual sera, and different Sm sera produce reactions which vary markedly in intensity and in
the extent to which they are distinct from the sharp precipitin line of Sm (Figure 4D).
Comparison with hemagglutination. Sera reactive
with each of the antigenic components of rabbit thymus
extract identified above, with the exception of anti-P-3,
were tested for anti-ENA antibodies by the hemagglutination technique with calf thymus extract-sensitized red
blood cells. As indicated in Table 2, only the sera reactive with RNP or Sm were identified by hemagglutination and, with the limited number of successive dilutions used, sera with antibodies to both RNP and Sm, as
determined by CIE, were identified by hemagglutination as reacting only with Sm.
Table 2. Comparison of ENA determination by CIE and hemagglutination
Patient
CIE result
Ha titer
GP
SA
MM
MS
AMF
BW
JO
RNP
Sm
Ha
P-2
“DNA”
RNP-2
RNP + Sm
1 :65,500 RNP
1:65,500 Sm
0
0
0
0
1:65,000Sm
ANTIBODIES TO SALINE ENA
103 1
Table 3. Diagnoses of ENA-positive patients reviewed*
ENA
reactivity
SLE
(75)
RNP, total
RNP only
Sm, total
Sm only
Ha
RNP-2
P-2
P-3
“DNA’
Unmatched
44
29
23
9
12
7
3
1
3
I
SLE/
PM
(6)
SLE/
PSS
(6)
3
2
3
2
0
0
0
0
1
0
5
1
3
0
PM
(5)
PSS
(4)
Misc.
(10)
4
3
0
0
3
3
0
0
0
0
0
0
0
7
2
2
I
I
0
0
1
0
4
0
1
1
0
0
0
0
1
0
0
0
1
1
* SLE = systemic lupus erythematosus; PM = polymyositis; PSS =
progressive systemic sclerosis; Misc. = miscellaneous.
Clinical correlations. Sufficient clinical information was available to substantiate the diagnosis in 106 of
the patients with antibodies to ENA. Seventy-five of
these patients had SLE and an additional 12 had SLE
with features of either polymyositis or scleroderma, 5
patients had polymyositis, 4 had scleroderma, 3 had undefinable connective tissue disorders, and 1 patient each
had tuberculosis, lymphoma, chronic active hepatitis,
essential mixed cryoglobulinemia, idiopathic Sjogren’s
syndrome, rheumatoid arthritis, and Takayasu’s arteritis. As may be seen in Table 3, antibodies to RNP were
the most common and the least diagnostically specific.
Antibodies to Sm were found in 29 SLE patients, in 2
patients with probable rheumatoid arthritis, and in a
patient with tuberculosis, antinuclear antibodies which
were possibly drug-induced, and no lupus-like manifestations. In view of the relatively small number of positive sera, the diagnostic specificity of antibodies to ENA
other than RNP and Sm is unclear.
A detailed analysis was performed of the clinical,
laboratory, and renal biopsy findings in the 87 SLE patients with and the 41 SLE patients without antibodies
to ENA whose medical records were sufficiently complete. As shown in Tables 4, 5, and 6, the various serologically defined groups were quite similar with respect
to the incidence of the major clinical and laboratory
features of SLE. Mortality was significantly increased in
our SLE patients with antibodies to Ha, and fewer patients with anti-Sm had high levels of antibodies to native DNA. Clinical features overlapping with other connective tissue disorders, i.e. Raynaud‘s phenomenon,
scleroderma-like skin changes, or myositis, were more
common in patients with anti-RNP, but the difference
in incidence did not’ reach the level of statistical significance. The increased incidence of overlap features
was significant only for the subgroup of patients not
listed in Table 4 with antibodies to both RNP and Sm
(P < 0.05). Finally, there was a tendency toward more
benign pathologic findings on light microscopic examination of renal biopsy specimens from patients with
anti-RNP, but the sample size was too small for the statistical test to be valid. Furthermore, the incidence of
clinically apparent renal abnormality and the incidence
of subendothelial electron-dense deposits in biopsy
specimens were not significantly different from those
found in SLE patients without anti-RNP.
DISCUSSION
The studies reported here indicate that the identification of antibodies to specific ENA, present alone or
in combination, can be conveniently carried out with
CIE by first establishing the enzyme sensitivity of the
reaction and then matching the reaction with those
formed by standard antisera to the limited number of
antigens with the same enzyme sensitivity profile. With
due attention paid to the artifacts created by overfilled
wells and non-immunologic reactions (Figure 4), CIE
has distinct advantages over the other available meth-
Table 4. Demographic and clinical features in patients with systemic lupus erythematosus correlated with ENA reactivity
Demographic features
Clinical features*
ENA
reactivity
No.
patients
Age, average
(range)
Years duration,
average (range)
Deaths
Negative
RNP, total
RNPonly
Sm, total
Sm only
Ha
RNP-2
41
52
32
29
11
12
8
38(13-60)
41 (18-78)
40(18-65)
41 (16-76)
39(21-54)
38(16-62)
41 (28-78)
9.0(2-23)
8.7(2-24)
9.3 (2-24)
7.8(2-21)
8.6 (2-20)
6.0(1-13)
11.3(420)
4(10)
5(10)
2 ( 6)
3(10)
0
4(33)j
0
* Number of patients; percentages shown in parentheses.
jP < 0.05.
CNS
Dermal Raynaud’s
Sicca
involvement vasculitis phenomenon syndrome
16(39)
18(35)
12(38)
13(45)
4(36)
6(50)
3 (38)
lO(24)
21 (40)
lO(31)
13(45)
6 (55)
8(67)
6(75)
I 1 (27)
25 (48)
14(44)
13(45)
4(36)
4(33)
3(38)
4(10)
4 ( 8)
0
1 ( 3)
0
1 ( 8)
l(13)
Myositis
4(10)
3 ( 6)
2(6)
3(10)
2(18)
0
0
Scleroderma
0
5(10)
1(3)
3(10)
0
0
l(13)
KEISER AND WEINSTEIN
1032
Table 5. Laboratory findings in patients with SLE correlated with ENA reactivity
Laboratory findings;
ENA
reactivity
No.
patients
CH50
580
(nl > 150)
AntinDNA
Platelets
40,000
Negative
RNP, total
RNP only
Sm, total
Sm only
Ha
RNP-2
41
52
32
29
11
12
8
22(54)
22(42)
14(44)
14(48)
3(29)
6(50)
5 (63)
28(68)
26(50)
16(50)
11 (38)t
2(18)
5 (42)
6(75)
15(37)
21 (40)
I 1 (34)
12(41)
2(18)
7(58)
3 (33)
Coombs
Proteinuria
(>350
mg/24 hr)
Azotemia
(Cr > 1.5
mg/dl)
11 (27)
24(46)
13 (41)
8(28)
2(18)
7(58)
4(50)
16(39)
21 (40)
13(41)
12(41)
3(27)
6(50)
3 (38)
lO(24)
14(27)
S(25)
7(24)
0
6(50)
2(25)
+
* Numbers of patients; percentages shown in parentheses.
t P < 0.05.
ods for the routine detection and identification of antibodies to ENA.
Anionic antigens, such as the major ENA, can be
detected with greater sensitivity by CIE than by Ouchterlony agarose gel immunodiffusion, because in CIE
antigen and antibody do not diffuse randomly but are
directed toward each other by the electrical field. With
appropriately spaced wells, the immunologic relationships among various antigens can be determined just as
in Ouchterlony immunodiffusion. Hemagglutination is
a.more sensitive technique than CIE and is more easily
quantitated. However, our results indicate that antibodies to antigens other than RNP and Sm which are
detectable in rabbit thymus extract and in a purified extract of rat liver chromatin by CIE are not detected by
hemagglutination with red cells sensitized with calf
thymus nuclear extract (Table 2). Furthermore, the
hemagglutination assay as conventionally performed
may fail to detect the presence of antibodies to RNP in
sera which also contain antibodies to Sm. This can occur because in the hemagglutination assay, antibodies to
RNP and Sm are distinguished by comparing the result
obtained from red cells coated with ribonuclease-treated
antigen to that obtained from red cells coated with untreated antigen; similar hemagglutination titers indicate
the presence of anti-Sm, and a much lower titer with ribonuclease-treated antigen indicates the presence of
anti-RNP. If serum dilutions are arbitrarily limited to
1:65,000 or 1:100,000, as is generally the case, rather
than continued to the several million-fold dilution often
necessary to reach a negative end point (9,the drop in
hemagglutination titer with ribonuclease-treated antigen indicative of the presence of anti-RNP will not be
evident should the serum also contain antibodies to Sm
in titers above the arbitrarily set level.
Our findings with respect to the enzyme sensitivities of major ENA are not entirely in accord with previous reports. We found Ha to be resistant to treatment
with trypsin, even when using a standard anti-Ha serum
provided to us by investigators who described Ha as
trypsin-sensitive (10). Different conditions of digestion
or our use of a chymotrypsin inhibitor-treated trypsin
preparation may be responsible for this discrepancy. Sm
has not previously been noted to be ribonuclease-sensi-
Table 6. Renal biopsy findings in patients with SLE correlated with ENA reactivity
Renal biopsy findings*
Electron microscopic
examination
Light microscopic examination
ENA
reactivity
No.
patients
biopsied
Negative
RNP, total
RNP only
Sm, total
Sm only
Ha
RNP-2
20
20
11
13
2
8
3
WNLor
mesangial
3 (15)
16 (30)
2(18)
4 (31)
0
(13)
2 (67)
Membranous +
Membranous proliferative
2 (10)
4 (20)
2 (18)
3 (23)
2 (100)
2 (25)
0
3 (15)
4 (20)
2 (18)
2(15)
0
2(25)
1 (33)
* Numbers of patients; percentages shown in parentheses.
Proliferative
12(60)
6(30)
5 (45)
4(31)
3 (38)
0
SubendoNo.
thelial
performed deposits
13
15
8
10
2
6
2
7(54)
4(27)
3 (38)
4(40)
1(50)
3 (50)
0
ANTIBODIES TO SALINE ENA
tive, but this may not have been appreciated, especially
in studies in which hemagglutination or complement
fixation is used, because nuclear extracts seem to contain at least two components with Sm reactivity, one
sensitive to the action of ribonuclease and one resistant.
Findings consistent with the existence of two Sm
components, and with the partial identity of one of
these components to RNP, have been observed previously, but these findings have been interpreted in different ways. Kurata and Tan found as we have that the
reaction of Sm and anti-Sm produced two precipitin
lines on CIE, but they attributed only the late-appearing diffuse line to Sm (1 1). Dorsch and coworkers, using
Ouchterlony immunodiffusion, identified sera reactive
only with Sm on the basis of their producing a single
precipitin line upon diffusion against nuclear extract
which persisted when ribonuclease-digested extract was
used and which showed a reaction of complete identity
with a standard anti-Sm serum. However, they noted
that this Sm-anti-Sm reaction spurred over the reaction
with nuclear extract of sera containing anti-RNP, indicating that there was a relationship of partial immunologic identity between the two systems. Dorsch et a1
concluded that anti-Sm sera must also contain antibodies to RNP (17). Mattioli and Reichlin had previously reported the same immunodiffusion findings,
but they offered a different explanation for the spurring
of Sm-anti-Sm over RNP-anti-RNP. On the basis of
complement fixation studies with absorbed sera, Mattioli and Reichlin concluded that RNP and Sm must exist in nuclear extracts as components of molecular complexes rather than as individual molecular species, that
Sm occurs both alone and in combination with RNP,
and that RNP occurs only in a complex with Sm (18).
The complex nature and relatedness of Sm and RNP
postulated by Mattioli and Reichh are supported by
the recent report of Lerner and Steitz that the immune
precipitates formed by 32Pand 3sS-methioninelabeled
nuclear extracts and antibodies to RNP and Sm contain
the same seven polypeptides associated with several
overlapping species of small nuclear RNA (19).
We believe that our findings on the reaction of
Sm and anti-Sm and the relationship between Sm and
RNP represent a confirmation by CIE of the conclusions reached by Mattioli and Reichlin (18). During
electrophoresis of nuclear extract and sera reactive with
Sm, antibodies to Sm meet and precipitate with the
RNP-Sm molecular complex to form the sharp, rapidly
developing, ribonuclease-sensitive precipitin line. Many
anti-Sm sera are of high titer (1 1) such that there is a
considerable excess of antibody present which may, in
1033
effect, diffuse through this precipitin line to meet and
precipitate with the molecular complexes containing Sm
only. This second ribonuclease-resistant precipitin line
of Sm is delayed and diffuse in its appearance because it
arises from subsequent diffusion rather than from the
initial electrophoresis. Thus, with many anti-Sm sera,
the conditions of CIE may permit the two molecular
species of Sm to produce separate precipitin reactions
even though only one is evident under the conditions of
Ouchterlony immunodiffusion.
Rabbit or calf thymus extracts, as well as extracts
of purified mammalian chromatin, clearly contain multiple antigens reactive with patients’ sera in addition to
RNP, Sm, and Ha. Since different ENA may have similar temperature and enzyme sensitivities, and since
these parameters may not be constant from laboratory
to laboratory, the only reliable way to determine the
specificity of an ENA-reactive serum is by establishing
its immunologic relationship to a standard serum (8).
Thus, it is likely that some or all of the new antigens
identified in this study are identical to nuclear or cytoplasmic antigens previously described by other investigators (20-23), but this can be sorted out only by reacting standard sera together against thymus extract by
immunodiffusion or CIE. Additional antigens not present in thymus extracts, some with specificity for different rheumatic diseases, have been found in cytoplasmic
extracts, in the nuclei of proliferating cells, and in nuclear extracts from a human B lymphocyte cell line
(8,24). One such antigen, Ro (SS-A), could not be detected in rabbit thymus extract by CIE. Use of a Wil,
extract in addition to thymus extract in testing patients’
sera by CIE could extend the advantages of this method
to the detecting and identification of other acidic antigens with different clinical specificities.
The incidence in SLE patients of antibodies to
RNP, Sm, and Ha found in this study (41%, 23%, and
9% respectively) is virtually identical to that reported by
Kurata and Tan (1 l), who also used CIE, and is substantially higher than that reported in studies with
Ouchterlony immunodiffusion or hemagglutination
(3,9,17,25,26). Such differences in incidence are to be
expected on the basis of the differences in sensitivity
and specificity of these methods as discussed above. Our
findings confirm previous reports that antibodies to Sm
occurred almost exclusively in patients with SLE,
whereas the ENA reactivity of sera from patients with
diseases other than SLE was most often due to antibodies to RNP (3,4,7). However, a review of the clinical,
laboratory, and renal biopsy manifestations of our SLE
patients did not clearly support previously suggested as-
KEISER AND WEINSTEIN
1034
sociations of ENA specificity and specific patterns of
disease expression (2-4). In particular, clinical-serologic
correlations such as the increased incidence of overlap
features, the low incidence of serious renal disease and
the better prognosis associated with antibodies to RNP
(2-4), the increased incidence of vasculitis and the decreased incidence of serious central nervous system and
renal disease reported in patients with anti-Sm (27,28),
and the high frequency of antibodies to Ha reported in
SLE patients with Sjogren’s syndrome (9) were either
not present in our SLE patients or were not statistically
significant. The clinical-serologic correlations in our patients which were statistically significant, the increased
mortality rate in patients with antibodies to Ha, and the
increased incidence of overlap features in patients with
antibodies to both Sm and RNP require independent
confirmation because of the relatively small number of
patients involved and the possibililty of unperceived selection bias. In view of recent reports of instances of
variation in titers of antibodies to ENA with time and
level of disease activity (9,23,29), it may be that these
contradictory results are due to the inappropriateness of
studies of single serum samples. Longitudinal studies
correlating antibody specificity and titers with disease
activity may be required to determine the clinical significance, if any, of the various ENA antibodies.
ACKNOWLEDGMENTS
We are grateful to Drs. H. R. Holman, P. Maddison,
and I. Listowsky for generously providing reference sera and
purified chromatin extract, to the physicians on the Einstein
faculty who provided access to their clinical records, to Dr.
D. M. Marcus for reviewing the manuscript, to Bernice Samuels for technical assistance, to Dorothy Vasco for secretarial
support, and to Dr. S. Wassertheil-Smolar and Susan Slagel
for assistance with the statistical analyses.
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ANTIBODIES TO SALINE ENA
1035
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Home Study Course
A home study course in Immunodeficiency Diseases will be presented by the University of Wisconsin School of Medicine, to be completed between September 1 and December 31, 1980. The
course is developed and presented by Sheldon Horowitz, MD, Associate Professor of Pediatrics,
Division of Immunology, and Member, lmmunobiology Research Center.
This program meets the criteria for 36 hours AMA credit. For further information write to Home
Study-CME, 481 WARF Building, 610 Walnut Street, Madison, WI 53706.
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