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Serum IgG and IgM Rheumatoid Factors by Solid Phase Radioimmunoassay.

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SERUM IgG AND IgM RHEUMATOID FACTORS BY
SOLID PHASE RADIOIMMUNOASSAY
A Comparison Between Adult and Juvenile Rheumatoid Arthritis
RICHARD WERNICK, JOSEPH J . LoSPALLUTO, CHESTER W. F I N K , and MORRIS ZIFF
In this report, solid phase radioimmunoassays
that specifically measure IgG rheumatoid factor (RF)
and IgM RF in absolute concentrations are described.
Polyclonal RF preparations were utilized as standards,
and as little as 40 nglml of IgG RF and I nglml of IgM
RF were detected. The IgG RF concentration of 26
seropositive rheumatoid sera was 439 f 755 pghl
(mean f 1 SD), a level 107 times that of normal controls
( P < 0.001). In contrast, levels for 49 children with
juvenile rheumatoid arthritis (JRA) were 6.1 f 3.7 pg/
ml for those with a plyarticular onset (JRA-Po), 27.3 f
113 pglml for the pauciarticular group (JRA-Pa), and
12.6 f 20.7 pglml for the group with a systemic onset
(JRA-S). None of these values differed significantly from
the value of 6.0 f 3.9 pglml measured in a juvenile
control group. The IgM RF level in adult rheumatoid
arthritis (RA) of 175 f 221 pg/ml was also significantly
elevated compared to controls (P < 0.001). In JRA,
however, mean levels of 1.4 & 2.0 pglml (JRA-Po), 2.8
f 8.3 p d m l (JRA-Pa), and 1.1 f 0.7 pglml (JRA-S)
were not elevated significantly above the value of 1.2 &
1.2 pg/ml measured in the juvenile control group.
From the Rheumatic Diseases Unit, Department of Internal
Medicine, Southwestern Medical School. The University of Texas
Health Science Center. Dallas, Texas.
Supported by USPHS National Research Service Award
AM07055 and Research Grant AM18505.
Richard Wernick, MD: Instructor in Internal Medicine;
Joseph J. LoSpalluto, PhD: Professor of Biochemistry; Chester W.
Fink, MD: Professor of Pediatrics; M o m s Ziff, PhD, MD: Professor
of Internal Medicine and Recipient. USPHS Research Center
Award.
Address reprint requests to M o m s Ziff, PhD, MD, Department of Internal Medicine, The University of Texas Health Science
Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235.
Submitted for publication September 29, 1980; accepted in
revised form May 6, 1981.
Arthritis and Rheumatism, Vol. 24, No. 12 (December 1981)
Hidden IgM R F was not found in 9 JRA sera tested.
These marked differences in RF levels provide another
indication that adult RA and JRA are distinct diseases.
Beginning with the work of Kunkel et a1 ( I ) ,
evidence has accumulated for the presence of IgG
complexes, with sedimentation coefficients between
11s and 18S, in the sera of patients with rheumatoid
arthritis (RA) (2,3). These intermediate complexes
consist of self-associated IgG rheumatoid factor (RF)
molecules (43). Attempts have been made to quantitate serum IgG R F directly (6-16), but, as pointcd out
previously (17-21), the methods employed have been
insensitive (6-14) or lacking in specificity (7-16). Using these methods, previous workers have detected
IgG R F in sera of children with juvenile rheumatoid
arthritis (JRA) (7,12-14) in amounts comparable to
those found in adult RA sera (7,14). On this basis, it
has been claimed that similar pathogenetic mechanisms may underlie both JRA and RA (12,131.
We have modified radioimmunoassays (RIA)
for IgG R F and IgM R F described by Carson et al t 17)
so that results can be expressed in absolute amounts,
and we demonstrate in this report that these modified
assays are both highly sensitive and specific for these
antiglobulin molecules. Application of the assays to
JRA sera revealed that, in contrast to adult RA, levels
of both IgG RF and IgM R F were normal for virtually
all juvenile patients tested.
PATIENTS AND METHODS
Subjects. Forty-nine patients with JRA, 26 with seropositive adult KA, 7 with juvenile systemic lupus erythemat o w s (SLE), and 5 with juvenile dermatomyositis (DM)were
studied. In addition, 46 normal adults, found to be seronega-
1502
tive by the sensitized sheep cell agglutination (SSCA) test,
and 32 children with scoliosis or chronic neurologic disease
were employed as control groups. RA patients met the
criteria for classic or definite disease (22). The diagnosis of
JRA was based upon the revised criteria of the American
Rheumatism Association (ARA) (23); 11 patients had a
polyarticular (JRA-Po), 29 a pauciarticular (JRA-Pa), and 9 a
systemic onset (JRA-S). SLE was diagnosed according to
the ARA criteria (24), and the diagnosis of DM was made by
standard clinical criteria. No patient had received either
gold, penicillamine, or antimalarial drugs in the preceding 6
months; none was receiving greater than 10 mg daily of
prednisone or its equivalent at the time of the study. Serum
was stored at -20°C until ready for use.
Preparation of immunoglobulins (Igs). Rabbit and
human IgG were prepared from Cohn Fraction I1 (Sigma
Chemical Co., St. Louis, MO) by chromatography on DEAE
cellulose in 0.01M sodium phosphate buffer, pH 7.5. Monoclonal IgM-kappa was purified from the serum of a patient
with Waldenstrom's macroglobulinemia by precipitation
with 33% ammonium sulfate and gel chromatography on a
Bio-Gel A-5m (Bio Rad Laboratories, Richmond, CA) column. Commercially obtained IgA (Calbiochem-Behring
Corp., La Jolla, CA) was further purified by repetitive
DEAE cellulose chromatography by using a stepwise sodium phosphate gradient at a constant pH of 7.5. Both IgA and
IgM were also subjected to protein A Sepharose (Pharmacia
Fine Chemicals, Piscataway, NJ) chromatography, and the
effluents were collected. All Igs were demonstrated to be
pure by double immunodiffusion by using antisera to specific
Ig and to whole human serum (Miles Laboratories, Costa
Mesa, CA). Preparations were concentrated by ultrafiltration by using an Amicon PM-I0 membrane (Amicon Corp.,
Lexington, MA). Protein concentrations were determined
spectrophotometrically; we assumed an optical density for a
1 mg/ml solution at 280 nm of 1.4 for IgG, 1.2 for IgA, and 1.1
for IgM.
Purification of Fab fragment of rabbit and human
IgG. Purified IgG was digested with 1% pepsin (w/w) for 22
hours at 37°C: in 0.1M acetate, pH 4.0. The resulting digest
was then fractionated over Sephadex G- 150 (Pharmacia) in
0.05M Tris, 0.2M NaCl buffer, pH 7.4. The F(ab')* fraction
was then pooled and dialyzed against 0.1M phosphate buffer,
pH 7.0, and digested with 3 mg papain per 100 mg F(ab')? for
16 hours at 37°C with 10 mM cysteine and 2 mM sodium
EDTA. This digest was applied to Sephadex G-100 (Pharmacia), and the first peak was collected and concentrated by
ultrafiltration. Double immunodiffusion produced precipitin
lines with antisera to Fab, but not to Fc. Protein concentrations of Fab and of the Jg fragments described below were
measured by Lowry's method (25).
Purification of human light chains. DEAE cellulose
purified human IgG was applied to a protein A Sepharose
column, eluted with 1M acetic acid, and dialyzed against
0.55M Tris HCI buffer, pH 8.2. The IgG was then reduced
and alkylated by a standard method (26), and a light chain
preparation was obtained by Sephadex G-100 gel chromatography in IN acetic acid. Precipitin lines formed in double
immunodiffusion gels with antiserum to light chains but not
with anti-Fc.
Immunoadsorbents. Purified IgA, IgM, IgG, Fab, and
WERNICK ET AL
light chains were coupled to Sepharose 4B (Pharmacia) by
the method of Cuatrecasas (27) after the Sepharose had been
activated with cyanogen bromide by the method of March et
a1 (28). Rabbit and human Cohn Fraction I1 were crosslinked with glutaraldehyde by the method of Avrameas (29).
Gels were stored at 4°C.
Xabbit anti-human IgG Fd. Commercial rabbit antiserum to human Fab (Calbiochem-Behring) was precipitated
with 40% ammonium sulfate. The precipitate was redissolved, dialyzed against 0.1M sodium acetate, pH 4.0, and
incubated with 1.5% pepsin at 37°C for 24 hours. The F(ab')*
fraction was isolated by Sephadex (3-150 chromatography
and then further purified by passage over a protein A
Sepharose column to remove undigested IgG. The effluent
was then applied to an Fab Sepharose immunoadsorbent
column, and anti-Fab was eluted with 0.2M glycine HCI, pH
2.8. This fraction was then dialyzed against Tris-saline and
affinity purified over IgM, IgA, and light chain Sepharose
columns.
Rabbit anti-human IgM. The F(ab'). fragment of the
IgG fraction of rabbit antiserum to human p chain (Calbiochem-Behring) was prepared as described for anti-Fd above
and then applied to an IgM Sepharose immunoadsorbent
column. Anti-IgM was eluted with 0 . 2 M glycine HCI and
dialyzed against Tris-saline. Further affinity purification was
then performed over IgG, IgA, and light chain Sepharose
columns.
Radioiodination of rabbit antibodies. F(ab')? fragments of rabbit IgG anti-human IgM and anti-human IgG Fd
were labeled with IZ5l by the lactoperoxidase method of
Marchalonis et a1 (30). Specific activity varied from 5002000 cpmtng protein.
Preparation of IgM RF standard. Serum obtained
from a patient with Felty's syndrome was heat inactivated at
56°C for 30 minutes and then precipitated with 40% saturated
ammonium sulfate. The pellet was redissolved in and dialyzed against 0.1M saline, and the retentate was incubated
on a glutaraldehyde cross-linked human Cohn Fraction I1
column equilibrated in Tris-saline, pH 7.4, for 1 hour at 37°C
and 16 hours at 4°C. The column was then eluted with 0.1M
glycine HCI, pH 2.8, and the eluate was rapidly neutralized
with 0. IM NaOH after filtration through a 0 . 4 5 ~Millipore
filter (Millipore Corp., Bedford, MA). This solution was then
chromatographed on a Sephadex (3-200 column equilibrated
in 0.2M sodium acetate buffer at pH 4.0. The first peak was
ultrafiltered, neutralized with 0.1M NaOH to pH 7.4, and
dialyzed against normal saline at 4°C. The concentration of
IgM was then measured by a solid phase RIA (described
below).
To determine the actual amount of measured IgM
retaining rheumatoid factor activity, an aliquot was readsorbed on glutaraldehyde cross-linked human Cohn Fraction
11. The immunoadsorbent, equilibrated in 0.1M phosphate,
0.25M NaCl buffer at pH 9.0, was incubated with the IgM R F
prepared above for 1 hour at 22"C, and then washed with the
equilibrating buffer. It was determined by RIA that 86% of
the IgM applied was bound to the column. The concentration
of IgM R F in the once-adsorbed preparation was then
calculated by multiplying its measured IgM concentration by
0.86. When control non-RF IgM was similarly treated, less
than 0.1% was bound after two adsorptions, indicating
SERUM IgG AND IgM RHEUMATOID FACTORS
Table 1. Measurement of normal IgG as IgG RF in the absence of
pepsin digestion*
IgM RF
detected,
Fdml
Pep NHSt
NHS + IgM RF
Pep (NHS + IgM RF)
Pep IgG$
IgG + IgM RFI
0
1.2
0
0
I .2
1gG KF
detected,
cpm bound
0
2113
0
0
2415
* Purified 1gM R F was added to IgG RF-free
normal human serum
at a concentration of 1.2 pglml. The solution was then tested at a
final dilution of 1:lOO for 1gG RF before and after pepsin digestion.
An equivalent amount of IgM RF was also added to a 12 mdml
solution of purified normal IgG and then tested for IgG RF. lo5 cpm
of '"I-anti-Fd were added in each experiment.
t Pepsin digested normal human serum (NHS).
t Pepsin digest of 12 mg/ml IgG in saline.
8 I2 mg/ml IgG and 1.2 Fg/ml IgM RF in saline.
negligible nonspecific binding. We chose not to adsorb and
elute the entire preparation of IgM RF a second time because
it has been shown that there is significant loss of RF activity
after 2 exposures to pH 2.8 (31), a phenomenon that we have
also observed.
To further assess the purity of the IgM RF preparation, its binding to human IgG-coated tubes was compared to
that of 2 monoclonal IgM RFs, which had been isolated by
Dr. P. Roberts-Thomson by gel chromatography after cryoprecipitation. Binding of the polyclonal IgM KF was slightly
greater at all concentrations assayed.
Preparation of IgC RF standard. An F(ab')? IgG RF
standard was prepared by precipitating the same serum used
for the isolation of IgM RF with 40% saturated ammonium
sulfate, dialyzing against 0.1M sodium acetate, pH 4.0, and
then digesting with 1.5% pepsin at 37°C for 22 hours. The
digest was centrifuged, and then the supernatant was neutralized with 1M Tris base, and incubated on a glutaraldehyde cross-linked rabbit Cohn Fraction I1 column, and
equilibrated in 0.05M Tris, 0.2N saline, pII 7.4, first at 37°C
for 1 hour, then at 4°C for 16 hours. The column was eluted
with 0.1M glycine HCI, pH 2.8, and the eluate was passed
through a 0 . 4 5 ~Millipore filter and rapidly neutralized with
0.1M NaOH to pH 7.4. This solution was then dialyzed
against normal saline at 4°C.
Because there might have been nonspecific adsorption of protein to the cross-linked rabbit IgG or loss of KF
activity during purification, an aliquot of the dialyzed solution was reincubated with the glutaraldehyde cross-linked
IgG under the conditions described above for IgM RF.
Ninety percent of the applied protein was bound. The
concentration of F(ab')z IgG RF in the dialyzed solution was
then calculated by multiplying the protein concentration by
0.90. Neither IgA nor IgM were detected in this preparation
by double immunodiffusion. Although IgM KF activity was
abolished by pepsin digestion (see below), it is conceivable
that minimal IgA RF contamination was present, though
most IgA is destroyed by pepsin digestion as well (6).
To assess directly nonspecific protein binding, pepsin-digested normal serum was incubated with the immun-
1503
oadsorbent according to the protocol described above. Less
than 0.3% of the initially applied protein was bound after the
second adsorption, again indicating minimal nonspecific
binding of non-RF protein.
Radioimmunoassay of total IgM. Microtiter wells
(Cooke Laboratory Products, Alexandria, VA) were coated
with 100 pI of a 1 pg/ml solution of F(ab')z rabbit anti-human
IgM in Tns-saline for 2 hours at room temperature, washed 3
times with Tris-saline. and then incubated with 1% bovine
serum albumin (BSA) in Tris-saline for 1 hour at room
temperature. Samples to be tested were diluted in I% BSA in
Tris-saline and incubate5 for 16 hours at 4°C. Wells were
then washed 5 times with buffer and 100 pl of a 200 ng/ml
solution of "'1 F(ab')z rabbit anti-lgM were added and
incubated at 4°C for an additional 24 hours. The wells were
again washed 5 times with buffer, cut out, placed in tubes,
and counted individually. Samples were tested in triplicate,
and a standard curve was developed using a normal human
serum with a known IgM concentration as determined by
single radial immunodiffusion (32).
Radioimmunoassay of IgG RF. To determine levels of
IgG RF, the procedure of Carson et a1 (17) was modified.
Sera were pepsin digested by diluting 50 pI in 100 pl of a 1
mg/ml solution of pepsin in 0. IM acetate, pH 3.6 (resulting in
a final pH of 4.1), and then incubated at 37°C for 22 hours.
After centrifugation at 2,500 rpm for 20 minutes, supernatants were diluted in l % BSA in Tris-saline and the pH
measured to ensure adequate neutralization. Falcon 12 x 75
mm polystyrene tubes (Falcon, Oxnard, CA) were coated for
2 hours at room temperature with 1 ml of a 30 pdml solution
of rabbit IgG in Tris-saline buffer, aspirated free of contents,
rinsed once with buffer, and then incubated for 1 hour at
room temperature with 1% BSA in Tris-saline. After aspiration, the neutralized serum pepsin digest was added and
allowed to remain at 4°C for 16 hours, after which the tubes
were aspirated and rinsed twice. One milliliter of a 0. I pg/ml
solution of "'1 F(ab')? rabbit anti-Fd in I % BSA in Trissaline was then added, and, after 24 hours at 4"C, the tubes
were aspirated, rinsed three times, and counted. All samples
were tested in duplicate and mean cpm bound computed.
Preliminary ammonium sulfate or polyethylene glycol precipitation before pepsin digestion did not alter the results
obtained, and, therefore, whole sera were tested. A standard
curve was prepared by assaying increasing amounts of
purified F(ab')? IgG RF added to pepsin-digested normal
human serum in triplicate tubes. For individual test sera.
cpm bound were converted to equivalent concentrations of
F(ab')2 IgG RF. These values were then multiplied by 1.5 to
obtain the equivalent concentration of intact IgG RF. Values
greater than 2 standard deviations above the mean of the
control group were defined as abnormal. Experiments demonstrating the need for pepsin digestion of test serum were
performed prior to the preparation of an IgG RF standard,
and the results were expressed as the difference in labeled
rabbit antibody cpm bound to the test solution and a BSA
control.
Radioimmunoassay of IgM RF. To determine levels
of IgM RF, the procedure of Carson et a1 (17) was modified.
Polystyrene tubes were coated with 30 pg of human IgG in 1
ml of Tris-saline for 2 hours at room temperature. After
aspiration of contents. the tubes were rinsed once with Tris-
WERNICK ET AL
Table 2. Specificity of rabbit antibody preparations*
Percent cpm bound
Ig coat
Anti-lgM
Anti-lgG Fd
Human lgGt
Human IgMS
Human IgAS
Light chains
Rabbit IgG
0.3
31.6
1.9
28.1
0.03
0.3
0. I
0.2
NDO
ND
* Polystyrene tubes were coated with an excess of the purified
immunoglobulin specified for 2 hours, quenched with BSA, and
incubated for 16 hours with either ’251-labeled rabbit anti-IgM or
anti-IgG Fd. After washing, tubes were counted.
t Tubes were coated with F(ab‘)2fragment for measurement of antiFd binding.
S Tubes were coated with neutralized pepsin digest for measurement of anti-Fd binding.
6 Not done.
saline, incubated with 1% BSA in Tris-saline for 1 hour at
room temperature, and aspirated. One milliliter of a dilution
of serum in 1% HSA in Tris-saline was then added, and the
tubes were incubated for 16 hours at 4°C. After aspiration,
they were rinsed twice with buffer, and 100 ng of the F(ab’h
fragment of lZ5Ianti-human IgM were then added and the
tubes incubated at 4°C for 24 hours. They were then aspirated, rinsed three times with Tris-saline, and counted. All
samples were tested in duplicate and mean cpm bound
computed. A standard curve was prepared by assaying in
triplicate increasing amounts of purified IgM RF added to
normal human serum. The cpm bound for the sera tested
were then converted to equivalent concentrations of IgM
RF. Values greater than 2 standard deviations above the
control group mean were defined as abnormal.
Measurement of hidden IgM RF. Nine JRA sera,
three from each subgroup classified according to type of
onset, were selected to test for hidden IgM RF, i.e., IgM RF
in which antigen binding sites were blocked by autologous
IgG (33). Six of these patients had active disease at the time
of study. Disease duration ranged from 1 to 12 years (mean
6.3), while age of onset varied from 2 to 13 years (mean 6.6).
All patients were white, and 7 were female.
Sera were fractionated on a Sephadex G-200 (Pharamacia) column equilibrated in 0.2M sodium acetate, pH 4.0.
First peaks were pooled, but distal trailing edges were
excluded to avoid IgG contamination. After neutralization
with 1M Tris base, these IgM-containing fractions were
assayed for IgM RF. To determine whether hidden IgM R F
was present, the amount of RF detected was compared with
that in the original whole serum.
Serum immunoglobulin levels. Serum levels of IgG
and IgM were determined by single radial immunodiffusion
(32) using Meloy Immunoplates (Meloy Laboratories, Inc.,
Springfield, VA).
Statistical analysis. The significance of the difference
between RA and control mean values was determined by the
Mann-Whitney U test. The Kruskal-Wallis nonparametric
test was applied to juvenile subgroups. The significance of
correlations was determined by the Spearman rank order
correlation matrix.
RESULTS
Requirement for pepsin digestion of test sera in
IgG RF radioimmunoassay. A normal human serum
containing neither IgM RF nor IgG RF and a preparation of purified non-RF IgG were each mixed with
purified polyclonal IgM RF to test the possibility that
normal IgG, not previously digested with pepsin,
could bind either directly to the IgG tube coat utilized
in the radioimmunoassay via Fc-Fc interaction or to
IgM RF bound to the IgG tube coat (Table 1). Under
such circumstances, normal IgG would be detected
falsely as IgG RF. When IgM RF was added to the
undigested serum or to IgG in a final concentration of
1.2 pg/ml, the amount of “IgG RF” detected increased
from 0 to 2,113 cpm and from 0 to 2,475 cpm,
respectively, indicating that normal IgG was being
falsely measured as IgG RF in each case. Pepsin
digestion of the solution of IgM RF in normal serum
abolished this false positivity.
Specificity of rabbit antibodies. The labeled rabbit anti-IgM and anti-IgG Fd were shown to be highly
specific for IgM and human IgG F(ab’)*, respectively,
IpMRF~ng/lnl)O
5
11
0
7.5
,
500
15
.(o
20
25
4ooo
4500
IPM ( W m l )
xxx)
Figure 1. Comparison of binding of IgM RF and normal IgM.
Purified IgM and IgM RF were tested by the IgM RF RIA for
binding to human IgG-coated tubes. The range of concentrations for
IgM was chosen to simulate that of sera. Final concentrations are
shown. Results are expressed as the percent of lZ5l cpm added that
were bound. Background binding was 0.2%.
1505
SERUM IgG AND IgM RHEUMATOID FACTORS
Figure 2. Comparison of binding of F(ab')2 IgG RF and normal
F(ab')? IgG. Purified F(ab')? IgG and F(ab')* IgG RF were tested by
the IgG RF RIA for binding to rabbit IgG-coated tubes. The range of
concentrations for F(ab'), IgG was chosen to simulate that of sera.
Final concentrations are shown. Results are expressed as the
percent of "'I cpm added that were bound. Background binding was
0.2%.
by both double immunodiffusion and by measurements
of binding to tubes coated with various Ig preparations
(Table 2). To test the rabbit anti-IgM, polystyrene
tubes were coated with an excess of either purified
human IgG, IgM, o r IgA for 2 hours, washed, and then
quenched with BSA. To determine the specificity of
the rabbit anti-Fd, tubes were coated with IgG F(ab'h,
neutralized pepsin digests of human IgM and IgA, light
chains, or rabbit IgG. One hundred nanograms of the
respective '251-labeled antibodies were then added and
the tubes incubated for an additional 16 hours at 4°C.
The tubes were then washed and counted. Significant
binding for anti-IgM was observed only to IgM-coated
tubes and for anti-Fd only to IgG F(ab')2-coated tubes.
The absence of binding of anti-Fd to either isolated
free light chains or to polyclonal IgA containing bound
light chains indicated specificity for the Fd region of
the IgG F(ab'), fragment.
Comparison of binding of normal and RF
immunoglobulin. Normal IgM and pepsin-digested
normal IgG demonstrated negligible binding to IgGcoated tubes over a range of concentrations that might
be present in test sera. In contrast, binding of IgM RF
and IgG R F preparations increased in a concentrationdependent fashion (Figures 1 and 2 ) . Moreover, as
little as 1 ng/ml of IgM R F reproducibly bound 0.70.9% of the cpm added above background, while as
little as 25-50 ng/ml of F(ab'): IgG R F reproducibly
bound 0.2-0.4% of the cpm added above background.
Specificity of RF binding. To determine whether
the measured antiglobulins were truly binding specifically to IgG and not to BSA or uncoated polystyrene,
the binding of 5 RA sera to tubes coated with BSA
alone o r BSA and IgG was compared (Table 3).
Without IgG coating, mean IgG F(ab')? binding fell
from 347 to less than 2 pg/ml, while mean IgM binding
decreased from 272 to less than 2 pg/ml, indicating that
binding was almost entirely to IgG.
In a second experiment, sera were tested in
tubes coated with BSA and either whole IgG or its Fab
fragment. Mean binding of IgG F(ab')? for 8 RA sera
l
rabbit Fab was
decreased from 957 to 3.4 ~ g / m when
substituted for IgG, while mean IgM binding for 18
sera to tubes coated with human Fab instead of IgG
fell from 366 to I .5 pg/ml. This result indicates that the
antiglobulins detected were binding predominantly to
the Fc region of IgG and were therefore true rheumatoid factors.
Inhibition of RF binding by fluid phase IgG or
IgG fragments. To determine whether soluble fluid
phase IgG could bind IgM R F and inhibit its binding to
the solid phase IgG, IgM RF was diluted in either
saline or in a normal serum and assayed. No difference
in solid phase binding was found. Possible inhibition of
binding by pepsin-digested IgG fragments in the IgG
R F assay was examined by performing gel fractionation of a pepsin-digested RA serum and then assaying
the pure F(ab')z fraction alone or with the addition of
varying amounts of lower molecular weight fragments.
Again, no significant inhibition was detected.
Table 3. Specificity of RF assays'
Tube coat
IgG t BSA
RSA
IgG F(ab')?bound,
CLidmIt
347
2
117
< 2.0
(n
=
IgM bound.
PlWt
5)$ 272 ? 156
C2.0
(n = 5 )
* RA sera were assayed for binding of IgG F(ab'), and IgM to tubes
coated with BSA alone or with BSA plus either IgG (30 kghnl) or
IgG Fab (20 @-id).
t Mean 2 SEM expressed as d m l serum.
t Number of sera tested.
WERNICK ET AL
1506
30001
0
8
0
i
88
(r
8
0
8
88
0
8
G
8
am
0
A
Figure 3. IgG R F levels in RA and juvenile rheumatic disease. Horizontal line indicates upper limit of normal
(mean t 2 SD).
Effect of freezing and thawing. Normal and RA
sera were repetitively frozen and thawed 4 times, and
an aliquot of serum was removed and kept at 4°C after
each thaw. IgG RF and IgM R F levels did not vary
significantly among the 4 aliquots.
IgG RF levels in patient sera. The IgG RF level
for the 26 seropositive adult RA sera was 439 ? 755
pg/ml (mean 5 SD), a value 107 times greater than the
normal adult control level of 4.1 ? 2.3 pg/ml ( P <
0.001) (Table 4). Twenty-five of the 26 RA sera had
abnormal levels of IgG RF (Figure 3).
IgG R F concentrations for the juvenile groups
were as follows: JRA-Po. 6.1 2 3.7 pg/ml; JRA-Pa,
27.3 2 113 p.g/ml; JRA-S, 12.6 2 20.7 p,g/ml; and
control, 6.0 2 3.9 pg/ml. It should be noted (Figure 3)
that the higher mean values for the JRA-Pa and JRA-S
groups are due to markedly increased levels found in
rare patients in these groups. In fact, the respective
median IgG RF levels were 5.3, 5.0, 6.0 pg/ml, and 5.7
pg/ml, respectively. Additionally, mean values for the
DM and SLE groups were 4.5 t 0.6 and 7.5 4.4 pgi
ml, respectively. There were no statistically significant
differences among the juvenile groups. There was also
no significant correlation between JRA IgG RF levels
and either IgM R F levels, erythrocyte sedimentation
rate (ESR), duration of disease, age at onset, or age at
*
time of study. It can be seen in Figure 3 that only 1 of
11 JRA-Po, 2 of 29 JRA-Pa, and 1 of 9 JRA-S patients
had abnormal levels. In addition, only 1 of 7 juvenile
SLE and none of the 5 juvenile DM patients had
abnormal concentrations of IgG-RE'.
IgM RF levels in patient sera. The IgM R F level
for the seropositive adult RA group was 175 2 221 pg/
ml, a value of 134 times the control level of 1.3 5 0.96
Table 4. Serum R F levels in RA and juvenile rheumatic diseases
IgG RF
IgM RF
Subjects (no.)*
kg/mlt
%
positive
RA (26)
Normal adults (46)
JRA-PO ( 1 I )
JRA-Pa (29)
JRA-S (9)
DM ( 5 )
SLE (7)
Juvenile controls
(32)
439 f 755
4.1 f 2.3
6.1 f 3.7
27.3 2 113
12.6 f 20.7
4.5 f 0.6
7.5 f 4.4
6.0 5 3.9
96
4
9
7
II
0
14
3
kg/ml
175 f 221
1.3 f 0.96
1.4 f 2.0
2.8 2 8.3
1.1 2 0.7
1.8 f 2.0
1.5 f 1.3
1.2 2 1.2
%
positive
100
4
9
3
0
20
0
6
* RA = adult rheumatoid arthritis; JRA-Po = polyarticular onset
juvenile arthritis; JRA-Pa = pauciarticular onset; JRA-S = systemic
onset; DM = juvenile dermatomyositis; SLE = juvenile systemic
lupus erythematosus.
t Mean f I standard deviation.
SERUM IgG AND IgM RHEUMATOID FACTORS
e
o
o
1507
r
a
0
I
RA
(26)
Adult
Controls
(46)
I
I
JRA
JRA
Polyarticular Pauciarticular
(29)
(14)
JRA
Systemic
(9)
Dermatomyositis ( a )
SLE ( 0 )
112)
-
Juvenile
Controls
(32)
Figure 4. IgM RF levels in RA and juvenile rheumatic disease. Horizontal line indicates upper limit of
normal (mean + 2 SD).
pg/ml (P < 0.001) (Table 4). All 26 RA sera had
abnormal levels, which were in all cases greater than
the highest control value (Figure 4).
IgM RF levels for the juvenile sera were as
follows: JRA-Po, 1.4 + 2.0 pg/ml; JRA-Pa, 2.8 t 8.3
p,g/ml; JRA-S, 1 . 1 2 0.7 pg/ml; and control, 1.2 ? 1.2
pg/ml (Table 4). There was no significant correlation
between JRA IgM RF levels and either ESR, duration
of disease, age at onset, or age at time of study. Values
for DM and SLE sera were 1.8 ? 2.0 pg/ml and 1.5 ?
1.3 pg/ml, respectively. N o statistically significant
differences were found between the various juvenile
groups. Only 1 patient from each of the JRA-Po, JRAPa, and DM groups had elevated levels of IgM RF.
Hidden IgM RF. It has been reported that IgM
RF may be masked in patients with JRA by the binding
of native IgG (34,35). To determine whether the binding of IgM RF to homologous IgG-coated tubes was
inhibited by autologous IgG, three sera from each JRA
group were chromatographed on Sephadex (3-200 at
pH 4.0 as described above in order to isolate an IgMcontaining fraction devoid of IgG. When the RF activity of the IgM fraction was assayed, in all cases slightly
less, rather than more, IgM RF was detected in the
IgM fraction than in the corresponding unfractionated
serum (Table 5 ) , a reflection of minor losses due to
fractionation and exclusion of the trailing edge of the
peak. Thus, no evidence was obtained for the presence
of hidden IgM RF in the nine sera examined.
Relationship between IgG RF and IgM RF in
RA. Figure 5 presents a plot of IgG RF versus IgM RF
for the group of 26 adult KA sera, demonstrating a
significant correlation between the two (r = 0.603, P <
Table 5. Absence of hidden IaM RF in JRA
IgM RF, kg/ml
Patient
Serum
IgM fraction
1
0.3
0.4
0.3
7
1.7
0.7
0.7
0.7
1.3
1 .o
I .4
8
9
2.4
3.8
1.2
I .5
2
3
4
5
6
0.1
0.6
0.6
0.7
WERNICK ET AL
1508
0
0
0
8
p- 6001
240
I
a
0
0
I
~ - , o,
0'
25
,
75
0
0
,
,
m
,
,
475
,
-,
,
J
m800
IgM RF (&nl)
Figure 5. Correlation of IgM R F levels with IgG RF levels in RA
sera. The correlation was significant (r = 0.603, P < 0.002).
0.002). It should be noted, however, that in a number
of instances, one class of R F was disproportionately
higher than the other. The mean concentration of IgG
R F was 2.5 times greater than the mean IgM R F
concentration.
Relationship between RF and total immunoglobulin in RA. Total IgM and IgG levels for 17 sera
randomly selected from the RA group were 3.4 2 2.3
mg/ml (mean k SD) and 22.3 2 7.9 mg/ml, respectively. A significant correlation was found between paired
IgM and IgM R F values (r = 0.575, P < 0.02), but not
between IgG and IgG R F concentrations (r = 0.335, P
< 0.20). The IgG R F level in these 17 sera was 644 ?
873 pg/ml and represented 2.9% of the total IgG, while
the IgM R F level was 203 2 246 pg/ml and accounted
for 6.0% of the total IgM. The fraction of IgG with R F
activity ranged up to 9.8%, while as much as 14% of
the IgM in one RA serum was RF.
DISCUSSION
In this report, we have described measurement
of IgG RF and IgM R F by solid phase RIAs that are
highly sensitive as well as specific. In addition, we
have been able to express the concentrations of these
antibodies in absolute amounts by using R F standards
prepared from a rheumatoid serum. Applying these
assays, we have revealed a marked disparity between
the consistently elevated levels of both RFs in seropositive adult RA and the normal levels in JRA.
Previous adsorption assays that determined absolute quantities of IgG R F (7-15) have lacked specificity, because they detected normal IgG as R F (1720). Solid phase RIAs that measure IgG R F have also
been described (17,36,37), but these too have certain
limitations. All express results in terms of heterologous labeled antibody bound rather than as absolute
quantities of RF. Second, binding of Ig other than IgG
R F may take place in these assays, allowing spuriously
high levels of IgG R F to be measured. For instance Fc
to Fc interaction of normal IgG may occur (18). Also,
failure to inactivate IgM R F by pepsin digestion, as in
the method of Hay et a1 (36), allows binding of normal
IgG to IgM R F bound to IgG-coated tubes, as we have
shown. In addition, bound IgM R F may react with the
Fc of the whole rabbit IgG anti-IgG antibody used in
this method, causing spuriously high values for IgG
RF. If reduction and alkylation are used to inactivate
IgM RF, enhanced nonspecific binding of normal
serum IgG to IgG-coated tubes occurs (37). There is3
in addition, significant binding of normal IgG when
tested at physiologic concentrations in this assay (37).
We have modified the assay described by Carson et al (17) to enable the expression of results as
actual concentrations of RF. Polyclonal R F standards
prepared from the serum of a patient with RA have
been used, since we thought that such standards might
be more appropriate than monoclonal RFs. Since
individual clones within the population of RFs in any
given serum may vary in their affinity for IgG, no
standard can be truly absolute. Nevertheless, Koopman has demonstrated binding curves nearly identical
to each other for several purified IgM RFs (38), and we
believe that the values obtained in the present study
are at least a close approximation. As little as 1 ng/ml
of IgM R F and 40 ng/ml of IgG R F are detectable in the
present assays.
We chose to employ rabbit IgG instead of
human IgG Fc as the antigen for binding IgG R F for
two reasons. First, Pope and McDuffy (39) have
shown that rabbit IgG is more sensitive than human
IgG Fc for the detection of IgG RF. Second, background binding with rabbit IgG was significantly lower
than with purified human Fc, thereby increasing the
validity of low values.
Preliminary pepsin digestion of serum before
assaying for IgG R F has several advantages. For IgM
RF-containing serum, digestion abolished the binding
of normal IgG that had occurred when undigested
SERUM IgG AND IgM RHEUMATOID FACTORS
serum was used. Because Fc regions of IgG are
destroyed, binding of normal IgG to bound RF of any
class is not possible, nor is direct binding of normal
IgG via nonimmunospecific Fc-Fc interaction with the
IgG-coated polystyrene (18). In addition, pepsin digestion frees complexed IgG R F molecules and thereby
ensures their capacity to react with solid phase IgG.
Finally, we have shown that binding of Ig in RA
sera to IgG-coated tubes was directed to the Fc
fragment of IgG, since binding could be abrogated by
substituting BSA or Fab for whole IgG in both the IgG
R F and the IgM R F assays. Therefore, binding was
truly of a rheumatoid factor type.
The marked specificity of our assays is reflected by the following results. First, the mean IgG R F
level of 4.1 pg/ml for the normal adult controls is far
lower than previously reported (14,40). Secondly, the
ratio between RA and normal serum levels of IgG R F
of 107 is over 20 times greater than that obtained with
previous adsorption techniques (7-1 1,40) and over
three-fold greater than that measured by previous
RIAs (16,17,37). Finally, there was no overlap between IgM R F levels in RA and control sera. In a
recent publication (38), a similar technique for measuring IgM R F was also found to be highly specific.
JKA and adult RA have been considered by
some to be the same disease affecting different age
groups. However, only 5-10% of children with JRA
are RF-positive by agglutination testing (41). In addition, the two groups have different immunogenetic
backgrounds. When compared to controls, RA patients have a significantly higher frequency of both
HLA-Dw4 (42) and HLA-DRw4 (43), whereas JRA
patients have a significantly lower frequency of these
histocompatibility antigens (44,45).
As others have suggested (18,20,21,46), prcviously reported abnormal levels of IgG RF in JRA sera
(7,12-14) may have been spuriously measured because
of the poor specificity of the methods employed. Our
finding of normal levels of IgG RF in JRA sera, in
contrast to those in adult RA, is consistent with the
known differences between RA and JRA. Using a
different RIA for IgG KF, Pope and McDuffy obtained
a similar result for a group of 6 children with JRA (37).
Although a single patient from each of the JRAPa and JRA-S groups had markedly elevated IgG R F
levels in the absence of elevated levels of IgM RF, our
results do not support the value of routine measurement of IgG R F as an aid in diagnosis of JRA as
suggested previously (12). In addition, there was no
correlation between the ESR and either IgG RF or IgM
1509
R F levels, consistent with the fact that RF values
remained in the normal range regardless of disease
activity. This has been the experience of others with
respect t o IgM R F (14,47,48).
The 29 children with a pauciarticular onset
comprised over half the JRA group. HLA typing had
been previously performed in 21 of these patients by
Dr. Peter Stastny, and only 5 were positive for R27. It
would appear, therefore, that our JRA population was
not inordinately diluted by patients with a potential for
spondylarthropathy. Only 1 of 5 patients in the entire
group who were known to have the DRw4 antigen had
a slightly elevated IgM R F level, and none had abnormal IgG R F concentrations.
Finally, we have attempted to measure concentrations of hidden IgM RF, i.e., IgM RF whose antigen
binding sites are blocked by autologous IgG (33).
While hidden IgG R F would be unmasked by the
pepsin digestion employed, it is possible that hidden
IgM K F could have escaped detection in our routine
assay. Although there is evidence against the presence
of hidden IgM R F in JRA (33,49), an extensive study
(35)has demonstrated its presence in over half of JRA
patients, particularly in those with active disease or
with a polyarticular or pauciarticular onset. However,
experiments in the present study, in which serum IgM
was separated from IgG by gel filtration at acid pH and
then tested for R F activity, revealed no hidden IgM
RF, even though 6 of the patients tested had either
active disease or such an onset.
For RA sera, we have demonstrated significant
correlations of IgM RF levels with both IgG RF and
total IgM, although several patients had disproportionate elevations of either class of RF. It is of interest that
a substantial fraction of the total Ig had R F activity,
ranging as high as 14% for IgM and 9.8% for IgG.
Finally, measured concentrations of IgG RF were
approximately 2.5 times as high as those of IgM RF,
although IgM has long been regarded as the predominant class of R F in rheumatoid sera.
In conclusion, we have measured both IgG RF
and IgM R F in absolute quantities in the serum of
adults with RA and children with JRA and other
rheumatic diseases. The solid phase RIAs employed
have been shown to be highly sensitive and to measure
specifically only IgG or IgM that possesses R F activity. While it has been acknowledged that most patients
with JRA lack agglutinating IgM RF, we have dernonstrated normal levels of IgM R F by an assay requiring
only a primary antigen-antibody interaction. More
importantly, in contrast to previous findings (7,12-141,
WERNICK ET AL
1510
IgG R F levels were also normal in JRA, regardless of
the type of onset. These results provide further evid e n c e that adult and juvenile rheumatoid arthritis are
unrelated diseases.
ACKNOWLEDGMENTS
The authors would like to thank Dale Edelbaum for
his excellent technical assistance; Jean Hattarki and the
personnel of Texas Scottish Rite Hospital for Crippled
Children for their help in obtaining sera; DeVonda Warren,
Mary Battles, and Brenda Guest for the typing of the
manuscript; and P. Slopoko for his constant support.
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