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Serum antiimmunoglobulins reactive with human and rabbit igg in rheumatoid arthritis and other conditions.

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120
SERUM ANTIIMMUNOGLOBULINS
REACTIVE WITH HUMAN AND
RABBIT IgG IN RHEUMATOID
ARTHRITIS AND OTHER
CONDITIONS
DIANA J. STANKAITIENE, ALFONSAS A. MATULIS, HENRIKAS GUOBYS,
and JANINA P. JUSENAITe
IgG, IgA, and IgM antiimmunoglobulins reactive
with human and rabbit IgG were measured in patients with
rheumatoid arthritis (RA), patients with rheumatic fever
or osteoarthritis, and normal individuals. Values o f all
antiimmunoglobulins were significantly evaluated in RA
patients as compared with other groups and depended
upon activity and stage of the disease. IgM antibodies with
specificity for human IgG predominated quantitatively
over others in sera of RA patients with high titers of RF,
whereas most o f those reactive with rabbit IgC in latex
negative or positive RA patients belonged to IgG class.
The reaction with human IgG included thermostable and
thermolabile IgM antiimmunoglobulins but in that with
rabbit IgG only thermostable antibodies were active.
A striking immunologic feature of rheumatoid
arthritis ( R A ) is an increased level of antiimmunoglobulins (rheumatoid factors) in serum and
synovial fluid of patients. Antiimmunoglobulin activity
From the Laboratory of Immunology and the Department of
Arthrology. Institute of Experimental and Clinical Medicine of the
Ministry of Health of the Lithuanian SSR.
Diane J. Stankaitiene, M.D.: Head of the Laboratory of
Immunology: Alfonsas A. Matulis. M.D.: Professor of Medicine.
Director of the Institute: Henrikas Guobys. M.D.: Head of the Department of Arthrology: Janina P. JuBinaite, M.D.: Senior Scientific
Worker of the Department of Arthrology.
Address reprint requests to Dr. D. StankaitienC. Laboratory
of Immunology. Institute of Experimental and Clinical Medicine of
the Ministry of Health o f the Lithuanian SSR. 232000 Vilnius.
K . Po7elos. 18. USSR.
Submitted for publication March 21. 1977: accepted April 20.
1977.
Arthritis and Rheumatism, Vol. 21, No. I (January-February 1978)
is found in all three major classes of immunoglobulins
However, findings reported by various authors
differ significantly both quantitatively and qualitatively.
Differences in results may be due to different immunoabsorption techniques and different origins of the antigen (human, horse, or rabbit IgG). Some authors have
shown the prevalence of IgG type antiimmunoglobulins
as compared with those of IgM and IgA types (2,3).
Furthermore, it is known that conventional tests for
rheumatoid factors depend mainly on antibodies of IgM
type, whereas positive tests with rabbit IgG are more
specific for RA than are tests with human IgG (6,7).
The purpose of this study was to compare levels
of serum IgG, IgA, and IgM antiimmunoglobulins
reactive with human and rabbit IgG in patients with
RA, in patients with other diseases, and in healthy persons, and to investigate some of their properties such as
thermostability.
(1-5).
MATERIALS AND METHODS
Source of Sera. Serum samples were obtained from 49
patients (7 males and 42 females) with classic or definite RA
ranging in age from I8 to 7 I years (mean age: 50 years). The
duration of disease activity varied from 1 to I8 years (average:
6.5 years). Disease activity was graded on the basis of the
clinical signs ofjoint inflammation, the erythrocyte sedimentation rate, complete blood count, serum protein electrophoresis, and other laboratory tests. Patients were classified
in three grades: severe activity (14 patients); moderate activity
(21 patients); and slight activity (I4patients). Disease activity
was considered severe if all laboratory tests were strongly
SERUM ANTIIMMUNOGLOBULINS
positive, the erythrocyte sedimentation rate was above 50
mm/hour according to Panchenkov, and clinical manifestations of joint inflammation were pronounced. Disease activity
was classified as moderate if laboratory tests were positive,
with erythrocyte sedimentation rate from 30 to 50 mm/hour,
and if obvious signs of active arthritis were noted. Activity was
considered slight if laboratory measurements and body temperature were within the normal limits without exudative occurrence in joints or if clinical manifestations were insignificant and inconstant.
The evolution of the disease was classified according to
ARA criteria. Nineteen patients were examined a t the fourth
stage of RA according to Steinbrocker et al. (8), 20 were a t the
third stage, and the remaining 10 patients at the second stage
of the disease.
Sera were also obtained from 13 patients with osteoarthritis, all over 40 years of age; from 13 patients with rheumatic fever, from 22 to 55 years of age; and from 12 normal
blood donors, ranging in age from 19 to 47 years. All sera were
stored at -30°C and centrifuged at 12,000 to 15,000 rpm
before use.
Insoluble Cross-Linked Proteins. The immunoadsorbents consisted of insoluble human or rabbit IgG obtained by column chromatography on DEAE sephadex-A-50
according to Baumstark (9) and Kulberg (lo), respectively.
Purity of the preparations was verified by immunoelectrophoresis against appropriate antisera. Proteins were
concentrated by ultrafiltration through Diaflo membrane PM
10. Insoluble cross-linked preparations were made by a modification of the method of Avrameas and Ternynck (1 I ) . One
gram of protein was dissolved in 20 ml of 0.1 M acetate buffer,
p H 4.4, then 6 ml of 2.5%aqueous glutaraldehyde were added
drop by drop with gentle stirring. The mixture was allowed to
stand for 10 hours a t room temperature.
The resultant gel was homogenized in a tissue grinder
by adding 5 volumes of 0.1 M phosphate buffer, pH 7.2, and
centrifuged at 1,000 g. A similar step was repeated twice with
0.1 M glycine-HCI buffer, pH 2.5, and then with 0.1 M glycine-NaCI buffer, pH 8.0. Finally, solid material was washed
with 0.01 M phosphate buffered saline (PBS), pH 7.4, until the
final wash had an absorbance of less than 0.005 at 280 M in a
I-cm light path.
Serum Immunoadsorption. Twenty-eight milligrams of
appropriate immunoadsorbents suspended in 1 ml of PBS
were incubated each with 1 ml of serum for 1 hour at 37°C and
then overnight at 4°C. Incubation at 37°C was performed with
periodic shaking (three to four times) of the reactants. After
incubation the complex was spun down and thoroughly
washed ten to twelve times with PBS at 4°C by centrifugation
at 2,000 rpm. Elution of antiimmunoglobulins was carried on
for 1 hour in cold 0.1 M glycine-HCI buffer, pH 2.5, by
constant shaking. The eluates were then neutralized with 0.5 N
NaOH.
Quantitative Estimation of Immunoglobulins in Eluates.
Immunoglobulin contents were estimated by the single radial
immunodiffusion technique of Mancini (12), using monospecific antisera to IgG, IgA, and IgM (Institute of Epidemiology and Microbiology of N. Gamaleya, Moscow). The
dilutions of antisera in ager plates ( 1 mm thickness) were
picked up using appropriate doubling dilutions of standard
serum with known immunoglobulin concentrations checked
121
according to W H O 67/97 Ig reference serum. Five wells (3 mm
in diameter) in each agar plate were filled with standards to
establish standard curves. For eluates and standard samples,
the wells were filled twice. Eluates from blank tubes with
insoluble antigen only were included in order to test for the
release of IgG from immunoadsorbent. These controls were
consistently negative.
It is proposed that concentration of immunoglobulins
be expressed in IU/ml but that the values be calculated in
terms of weight based on the best available estimates (13). I n
order to facilitate comparison of our results with data obtained by other authors, we have used the conversion figure
obtained by Rowe et a1 (14), ie, 1 unit of activity of IgG
corresponds to 80.4 pg of isolated IgG; 1 unit of IgA to 14.2pg
of isolated IgA, and 1 unit of IgM to 8.47 pg of isolated IgM.
As little as 10 pg/ml of IgG and IgA, and 15 pcg/ml of IgM
could be detected in our assay.
Test for Thermostability of Antiimmunoglobulins. To
solve the question of whether IgG, IgA, and IgM antiimmunoglobulins reactive with human and rabbit IgG are
thermostable or thermolabile, parallel testing was performed
on sera heat-inactivated in a water bath for 30 minutes at 56°C
and noninactivated serum samples.
RESULTS
All sera tested, including those from normal persons, had antiimmunoglobulins of IgG class reactive
with both human and rabbit IgG (Table I ) . Their mean
values in eluates from R A sera were 42.4 f 4.1 and 44.4
f 1.9 pg/ml (range: 20 to 155 pglml and 20 to 105
pg/ml), to human and rabbit IgG, respectively. The
concentration of IgG antiimmunoglobulins reactive
with human and rabbit IgG in eluates from sera of
patients with rheumatic fever was not more than 39 and
46 pg/ml. Mean values of antibodies mentioned above
for normals were 24.0 f 0.4 and 22.5 f 2.4 pg/ml. The
concentration of IgG antiimmunoglobulins in eluates
from patients with osteoarthritis ranged from 20 to 31
pg/ml and from 22 to 38 pg/ml, respectively. for antiimmunoglobulins to human and rabbit IgG.
Antiimmunoglobulins of the IgA class with specificity of human IgG were found in 75.5% of R A patients with a mean value of 13.4 f 1.3 pg/ml (range:
< 10 to 39 pg/mI). The concentration of those reactive
with rabbit IgG ranged from < 10 to 29 pg/ml (mean
value 10.9 f 1.1 pg/ml). IgA antiimmunoglobulins were
detected in 69.4% of RA patients, but were not found
in sera of normal individuals or of patients with osteoarthritis. In eluates from sera of patients with rheumatic
fever they were found in only 30.7% and 23.1% of cases
for human and rabbit IgG, respectively (mean values:
4.1 f 1.3 and 3.1 f 1.1 pg/ml).
IgM antiimmunoglobulins to human IgG were
STANKAITIENE ET AL
122
Table 1. Concentration of IgG, IgA. and IgM Anti-Immunoglobulins Reactive with Human and Rabbit IgG in Eluates from Normal Persons and
Patients with Rheumatoid Arthritis, Rheumatic Fever, and Osteoarthritis ( p g / m l )
No.*
Groups
"lot
Rheumatoid
arthritis(n = 49)
Rheumatic fever
(n = 13)
Osteoarthritis
( n = 13)
Normal persons
(n = 12)
49
100
13
100
13
100
12
100
Human
IgG
No.
42.4 f 4.1
20-155
28.1 f 1 . 1
22-39
24.7 f 1.4
20-3 I
24.0 f 0.4
22-26
49
100
13
100
13
9i
I00
12
I00
kM
IgA
Mean f SE
Range
kG
Mean f S E
Range
Rabbit
IgG
No.
46
44.4 f 1.9
20- 105
28.2 f 1.8
23-46
24.4 f 0.6
22-38
22.5 f 2.4
1 1-27
* Number of positive cases.
t Percentage of positive cases. Amounts of antiimmunoglobulins
Human
I gG
No.
9i
Mean f S E
Range
Rabbit
IgG
37
75.5
4
30.7
13.4 f 1.3
<lo-39
4.lf1.3
<10-13
-
-
-
-
-
-
-
-
34
69.4
3
23.1
10.9 f 1 . 1
<lo-29
3.lf1.1
<10-10
No.
9i
Human
IgG
54.2 f 6.9
43
87.7 < I S 2 3 0
9.2f3.5
5
38.5 <15-35
3.8 f 1.5
3
23.1 <15-18
I
2.2 f 1.2
8.3 < I S 1 6
No.
37
29.7 f 3.6
75.5 < I S 1 1 8
2
2.8fI.I
15.4 < I S 1 9
3
4.1 f 1.6
23.1 <15-19
-
below the limits of sensitivity of the test were regarded to be equal
detected in 87.7% of RA patients with a mean value of
54.2 f 6.9 pg/ml (range: < 15 to 230 pg/mI), whereas a
mean of those reactive with rabbit IgG was only 29.7 f
3.6 pg/ml (range: < 15 to 118 pg/ml). Anti-rabbit IgG
were found in detectable amounts 75.5% of RA patients.
Mean values of IgM antiimmunoglobulins of both specificities for patients of two other groups were considerably lower than for RA patients. IgM antibodies reactive with rabbit IgG in eluates from sera of normals were
not detected. The difference in mean of antiimmunoglobulins of all three classes reactive with both
human and rabbit IgG between RA patients and normals or patients with osteoarthritis and rheumatic fever
was statistically highly significant (P < 0.001 ).
Our results (Table 2 ) showed that antiimmunoglobulins belonging to IgG and IgA classes increased independently of the total serum immunoglobulin level of appropriate class, (r = 0.136 f 0.144, P
> 0.05 and r = 0.257 f 0.141, P > 0.05, respectively),
though in a number of instances there was a relation
between elevated levels of IgM antiimmunoglobulins
reactive with human IgG in eluates of sera from RA
patients and increased concentration of total serum IgM
(r = 0.374 f 0.135, P < 0.01). A high IgM antiimmunoglobulin level was usually associated with an
elevated level of IgG antiimmunoglobulins to human
IgG, although no linear relationship existed between
their concentrations. When human cross-linked IgG was
used as a substrate, antiimmunoglobulins of the IgM
Rabbit
IgG
9i
-
to
I pg/ml,
class predominated quantitatively over those of the IgG
class in about two-thirds of eluates from RA patients.
The concentration of IgM antiimmunoglobulins correlated directly with the elevation of R F titers in latex
tube test (r = 0.655 f 0.110, P < 0.001). On the contrary, most of the antiimmunoglobulins reactive with
rabbit IgG, detectable in eluates of sera from latexpositive or latex-negative RA patients, belonged to IgG
class.
Increased levels of IgG, IgA, and IgM antiimmunoglobulins reactive with human and/or rabbit
IgG were associated to some degree with the progression
of RA. Values of IgG, IgA, and IgM antiimmunoglobulins reactive with human IgG were significantly
higher in patients at the third (P < 0.05) and fourth
(P < 0.001) stage of the disease as compared with patients at the second stage. Mean values of antiimunoglobulins reactive with human IgG were: for IgG antiimmunoglobulins-22.9 f 4.1, 45.4 f 5.9, and 46.7
f 3 pg/ml; for IgA-5.5 f 2.4, 14.9 f 2.4, and 14.6
f 2.2 pg/ml; for IgM--13.8 f 4.5, 54.6 f 8.3, and 64.5
f 14.7 pg/ml, respectively, for patients at the second,
third, and fourth stages of the disease.
However, no relation was found between the levels of IgG and IgA antiimmunoglobulins reactive with
rabbit IgG and the stage of the disease. Only IgM antiimmunoglobulins with specificity to rabbit IgG had
clearcut tendency to increase with the progression of the
disease, as indicated by their mean level in patients at
SERUM ANTIIMMUNOGLOBULINS
123
Table 2. Particulars of R A Patients
Anti-Immunoglobulins h g / m l ) Reactive with
Human IgG
Age
Patient (years)
TL
KV
KC
GR
AT
68
57
SA
71
45
53
66
40
33
45
57
63
54
66
66
so
zo
BG
LM
RA
BN
BB
JK
BA
so
JN
MP
SI
TR
GV
DN
GM
SA
DM
VD
RS
sz
DB
SH
cs
AB
GZ
JB
MO
ZE
A0
VK
VD
RA
ZJ
MG
VA
AA
RA
TM
sv
DE
ZR
Sex
F
F
64
M
53
54
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
M
F
F
F
F
F
F
F
F
F
F
F
F
F
F
M
M
F
F
F
M
M
M
F
F
F
F
51
64
44
64
59
51
50
62
34
53
63
63
52
49
42
66
46
47
31
52
61
20
33
62
19
18
18
35
62
32
22
46
Stage
of
Disease
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
111
Ill
111
111
111
111
111
111
111
111
111
III
111
111
Ill
Ill
111
Ill
Ill
111
II
II
11
II
II
II
II
II
II
II
Titer of R F
Latex-Tube Test
Rabbit IgG
Disease
Activity
IgG
IgA
IgM
IgG
IgA
IgM
Severe
Severe
Severe
Severe
Severe
Severe
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Slight
Slight
Slight
Slight
Slight
Severe
Severe
Severe
Severe
Severe
Severe
Severe
Severe
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Slight
Slight
Slight
Slight
Slight
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Slight
Slight
Slight
Slight
I20
60
20
26
I55
45
58
24
24
31
48
38
32
24
28
41
58
23
31
54
103
28
54
54
28
78
120
54
26
36
24
28
54
33
26
33
24
28
24
58
38
20
24
24
29
24
31
20
24
22
15
20
12
16
24
34
0
13
I1
16
10
10
17
0
22
12
15
10
16
39
17
10
25
22
15
32
10
12
18
0
12
22
13
20
0
0
0
15
0
230
48
33
64
220
51
135
0
0
18
57
35
23
65
33
100
38
35
41
100
135
87
36
38
64
80
57
43
73
48
43
58
I05
39
58
36
58
62
32
39
20
28
33
41
39
36
58
39
58
53
39
48
41
22
38
53
36
43
43
43
53
43
40
36
38
35
39
14
20
15
15
20
14
29
0
0
12
13
14
10
0
0
14
0
0
I2
12
29
20
16
14
20
16
0
10
13
12
0
17
18
18
15
0
0
10
10
10
24
0
0
10
10
14
0
0
0
62
15
19
38
70
42
I18
0
46
19
26
32
0
34
0
40
48
28
22
38
115
40
32
28
52
35
30
30
32
53
19
33
53
35
0
0
0
19
0
26
32
0
0
20
26
0
28
0
0
11
0
0
0
12
22
10
0
0
64
135
46
33
100
20
33
0
0
23
0
46
46
38
35
23
40
0
20
51
33
60
47
54
38
36
43
38
39
39
35
Human
IgG
Rabbit
IgG
1:2560
1 : 1280
]:I60
ND*
I : I280
1:640
]:I60
1:640
]:I60
1:640
1:2560
]:I60
]:I280
-*
-
-
1:640
]:I60
1:40
1:640
1:80
]:I280
]:I60
1:320
1:640
1:640
-
I :40
-
]:I60
1:80
ND
ND
-
ND
1:40
1.80
1:80
ND
1:320
]:I280
]:I280
1:640
1:640
1:640
1:80
1.80
1:320
]:I60
1:320
1:320
1:640
1:2560
1:640
]:I280
1:2560
1:640
I :40
I-
-
I:160
]:I280
1 :80
1:320
1:320
1:80
ND
1:640
1:80
1:320
]:I60
1:320
1:320
1:80
-
1:40
]:I60
-
1 :40
-
-
1:640
1:320
1:80
1:80
1:80
]:I60
-
-
-
Concentration of Serum
Immunoglobulins
(mg/100 ml)
IgG
IgA
IgM
I579
I800
I995
2520
2220
1080
1680
1350
I857
2565
I650
2775
1050
I005
1650
2100
I995
1470
2280
2100
I575
1500
I980
3240
2790
2070
I920
I155
1725
I875
1470
I740
2955
1440
I350
2280
I365
1995
I92
350
3 10
290
280
290
670
216
332
440
366
250
72
224
360
280
140
240
440
I88
188
366
360
324
326
184
320
I28
294
I50
I22
210
326
320
302
I84
270
500
246
310
266
70
200
310
230
294
212
I56
266
376
360
I02
1800
I690
I725
2775
I130
I690
I290
1320
1800
I320
1470
-
N D = nondetermined:
= negative.
Mean values of total serum immunoglobulins for normal individuals (n
=
59): IgG 1147 f 22, IgA 196.5 f 6.9. IgM 122.7 f 6.5 mg/100 ml.
164
484
260
260
230
I20
240
200
I80
I50
224
230
210
I92
I50
240
880
168
240
I36
I48
350
136
240
I28
230
460
I40
172
200
I72
230
120
300
I38
I20
240
110
178
I68
I80
I60
196,
I I6
I 92
I38
,
STANKAITIENE ET AL
124
MODERATE
VERY ACTIVE
~~
0
140
120
-
.
.
.
0
100-
80 *
.
.
0
60
-
40 -
0
0.00
?:
00000
:
0.0
800
-..
0
20
-
t
mt
substrate. There were no significant differences in their
means, except for the means of the antibodies reactive
with rabbit IgG in patients with severe and slight activity of RA (P < 0.01).
Dependence of IgA antiimmunoglobulin levels
upon the degree of activity of RA is similarly presented
in Figure 2. Mean levels of IgA were: for the patients
with severe activity of the disease-18.8 f 2.0 and 16.9
f 1.2 pg/ml, for the patients with moderate activity11.5 += 2.0 and 10.8 f 1.7 pg/ml, and for those with
slight activity-9.9 f 2.5 and 5.1 f 1.6 pg/ml for antibodies to human and rabbit IgG, respectively. The difference between their means in patients with slight
activity ( P < 0.02) and moderate or severe activity
( P < 0.001) was highly significant.
IgM antiimmunoglobulin levels in sera of RA pa-
MODERATE
SLIGHT
.
'ERY ACTIVE
"
o
r
000
.
800
..
n
00
.O
I
Figure 1. Serum IgG antiimmunoglobulins in rheumatoid arthritis patients: correlation with disease activity. Each point represents a determination from an individual patient's serum. = reactive with human IgG.
0 = reactive with rabbit IgG.
the second stage of the disease: 13.2 f 3.5 pg/ml, in
patients at the third stage-32.2 f 7.1 pg/ml, and in
patients at the fourth stage-35.2 f 7.5 pg/ml. Their
means in patients at the second stage of disease were
significantly lower as compared with patients at the
third ( P < 0.05) and fourth ( P < 0.02) stages of the
disease.
Levels of IgG antiimmunoglobulins in eluates of
sera from RA patients with different activity of pathologic process are shown in Figure 1. The mean values of
these antiimmunoglobulins were: for the patients with
severe activity of the disease-61.1 f 16.7 and 5 I .7 f
5.2 pg/ml, for those with moderate activity-39.2 f 4.8
and 44.5 f 2.3 pg/mI, and for the patients with slight
activity of RA-29.1 f 2.6 and 36.9 f 0.9 pg/ml, respectively, when human and rabbit IgG was used as a
0
..
..
.
0.
0
0
.
0
..
00
0
0
00
0
0. 00
0.0 00
0
0. 0000
....mo
OOOQOOOOO
..
0.
0000
..oO
..00
.
000
00
0.
00
0.H..
0
n
0000000
Figure 2. Serum IgA antiimunoglobulins in rheumatoid arthritis patients:
correlation with disease activity. Each point represents a determination
from an individual patient's serum. = reactive with human IgG. 0 =
reactive with rabbit IgG.
SERUM ANTIIMMUNOGLOBULINS
~~
~~
~~~
M O D E R A T E VERY ACTIVE
240
0
220 -
200
-
180
-
0
140 -
160
125
Parallel testing of single serum samples from 25
RA patients revealed that the antiimmunoglobulins of
all three classes reactive with rabbit IgG were thermostable, because the heating of sera at 56°C for 30 minutes prior to the adding of crosslinked rabbit IgG caused
no changes in their amounts, as detected in eluates by
immunodiffusion reaction. Antiimmunoglobulins of
IgG and IgA classes with specificity to human IgG were
also thermostable, but the great part of IgM antiimmunoglobulins reactive with human IgG was thermolabile. After heating, some sera lost their activity; in
other sera the antibody activity diminished by variable
degrees and was not altered in only 4 sera of 25 with
small or moderate amounts of IgM antiimmunoglobulins (Figure 4).
0
120 -
DISCUSSION
0
100
-
80
-
0
0
0
0
0 0
0
" 0
0
0
40
o0
- 0
Q)
0
0
it
0
0
0 0
0
0
Figure 3. Serum IgM anriimmunoglobulins in rheumaroid arthritis patients: correlation with disease activity. Each point represents a determination from an individual patient's serum. = reactive with human IgG.
0 = reactive with rabbit IgG.
tients with different degrees of activity of the disease are
depicted in Figure 3. The means of these antibodies
were: 87.6 f 17.3 and 44.1 f 6.7 pg/ml for the patients
with severe activity of RA, 47.7 f 7.9 and 30.2 f 5.5
pg/ml for the patients with moderate activity, and 31.0
f 7.5 and 14.6 f 4.7 hg/ml for the patients with slight
activity of the disease, for IgM antiimmunoglobulins
reactive with human and rabbit IgG, respectively. The
mean values of IgM antiimmunoglobulins with specificity for human IgG in patients with slight activity of
the disease were significantly lower as compared with
the values for patients with moderate (P < 0.05) and
severe (P < 0.01) activity. The mean value of those
reactive with rabbit IgG in patients with severe activity
of RA was also higher than that in patients with slight
activity (P < 0.002).
Our results have shown the presence of IgG, IgA,
and IgM antiimmunoglobulins in increased amounts in
sera of RA patients. All sera, whether from normal individuals or patients with RA, osteoarthritis, or rheumatic
fever, contained IgG antiimmunoglobulins, although
the level in RA patients was significantly higher. However no relation was found between the concentration of
IgG antiimmunoglobulins in eluates from sera and total
serum IgG level in RA patients. High concentration of
IgG antiimunoglobulins does not reflect an elevation
of serum IgG level because its value is influenced by a
concentration of various antibodies of a different nature, as a majority of them belong just to this class. On
the other hand, a high concentration of IgG antiimmunoglobulins suggests that the immunosorbent used
does not result in a nonspecific binding of proteins onto
it caused by hypergammaglobulinemia. When insoluble
human IgG was used as a substrate, IgM antiimmunoglobulins predominated quantitatively over antiimmunoglobulins of other classes in eluates from sera of
RA patients with high titers of RF, revealed with human
IgG-coated latex.
By using rabbit IgG as antigen, the amounts of
antiimmunoglobulins of IgG class, demonstrable in
eluates, exceeded those of IgM class in most cases of
both seropositive and seronegative RA. The lower titers
of RF, and less often positive latex-agglutination reactions revealed by using rabbit IgG as antigen in comparison with human IgG, probably result in some
way from the fact that in agglutination reaction, antibodies of IgG type are less active than those of IgM type
(1 5 ) because in our assay most antiimmunoglobulins
reactive with rabbit IgG belong to the IgG class. Moreover, the finding of elevated levels of IgG anti-
STANKAITIENE ET AL
I26
220
240
1
20
40
60
80
100
120
140
160
HEAT-INACTIVATED SERUM
180
200 JgM
rus/m'
Figure 4. Heat-labile and heat-stable IgM antiimmunoglobulins reactive with human IgG in eluates of sera
from rheumatoid arthritis patients: comparison of values obtained with single serum nonheated and heated
at 56'C for 30 minutes.
immunoglobulins reactive with human and/or rabbit
IgG may be a diagnostic aid in early stages of the disease
and in differential diagnosis of difficult cases, as indicated by others (3,16).
IgA antiimmunoglobulins in eluates from sera of
RA patients were detectable only in a low concentration. Within the limits of the sensitivity of the test employed, IgA factors were demonstrable neither in normal sera tested nor in the sera of osteoarthritis patients.
Small amounts of IgA antiimmunoglobulins found in
eluates from RA patients and a failure to detect them in
osteoarthritis patients in our assay may be explained by
findings (17) that in a number of instances antibodies of
IgA class are not detectable by precipitation reaction. In
our assay IgA antiimmunoglobulins were revealed in
eluates from sera just by means of precipitation reaction
in agar gel with appropriate antiserum (immunodiffusion).
The literature contains conflicting data concerning values of anti-IgG antibodies in RA patients
and normals. The findings in RA patients presented here
are similar to those obtained by Torrigiani and Roitt
SERUM ANTIIMMUNOGLOBULINS
( l ) , who used rabbit IgG as antigen, with the exception
of lower IgA antiimmunoglobulin levels revealed by us.
Vischer and BHnziger (4)using insoluble human IgG
were unable to detect IgA antiimmunoglobulins in
measurable amounts in eluates not only from normal
individuals’ sera but also from sera of RA patients.
However, using human IgG as a substrate, some authors
(2,3) have found IgA antiimmunoglobulins in considerable amounts in eluates from sera of RA patients and
even of normals. Values for IgG antiimmunoglobulins
in seropositive patients reported by these authors were
significantly higher than those for IgM antiimmunoglobulins, which contrasts with our data.
We noticed that the presence and the level of all
antiimmunoglobulins tested showed a certain relation to
the disease activity as well as to its evolution. IgM
antiimmunoglobulins of both specificities were mostly
related to the severe activity and late stage of RA.
Few data in the literature concern studies of
species specificity of anti-IgG antibodies of separate
classes in RA. Both homologous and heterologous gammaglobulins (IgG) were used as substrates in determinations only of IgG antiimmunoglobulins but not of IgA
and IgM classes in adult RA patients. Thus Torrigiani
and Roitt ( I 5 ) detected in nearly every instance a higher
concentration of IgG antiimmunoglobulins in the sera
of RA patients than in rabbit sera, using a crosslinked
preparation of horse gammaglobulin. Values of IgG
antiimmunoglobulin reactive with both human and
rabbit IgG reported by Ilter and Turner (18) in RA patients and normals were somewhat lower than those
obtained by us. They also found that the sera tested
tended to have greater reactivity with human than with
rabbit IgG. Florin-Christensen et al. ( 5 ) have shown
that in juvenile RA a disparity in immunoglobulin class
and in concentration of antiimmunoglobulins was often
obtained from a single serum with human and horse
gammaglobulins; the reaction with human FII was
expressed in considerably greater extent. This disparity
possibly related to different specificities or affinities
of antibodies. In our assay the mean values of IgG antiimmunoglobulins with specificity to human or rabbit
IgG were very similar (42.4 f 4.1 and 44.4 f 1.4 grams),
though some sera of RA patients exhibited reactivity
to a greater extent with human and some sera with
rabbit IgG. The findings reported by Skalba and Stanworth (19) suggest that rheumatoid sera contain rheumatoid factors capable of recognizing structural similarities and differences between IgG of different mammalian species.
In a separate study we also investigated the ther-
127
mostability of antiimmunoglobulins reactive with human and rabbit IgG. In the experiments of the authors
mentioned previously only native but not heat-inactivated sera were examined. By parallel testing of
heat-inactivated and noninactivated sera we have
shown that all antiimmunoglobulins of the main classes,
reactive with rabbit IgG were thermostable, whereas
among IgM antiimmunoglobulins reactive with human
IgG there were both thermostable and thermolabile
antibodies. The concentration of thermolabile factors
increases together with that of thermostable ones. Thus,
antiimmunoglobulins different from rheumatoid factors
are also present in increased amounts in sera of RA
patients.
Our data have revealed some differences in the
nature of antiimmunoglobulins reactive both with human and rabbit IgG. In order to improve laboratory
diagnosis of RA. we propose to use in parallel both
human and rabbit IgG for determination of antiimmunoglobulins in patients’ sera.
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patients with rheumatoid arthritis and normal subjects.
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2. Panush RS, Bianco NE, Schur PH: Serum and synovial
fluid IgG, IgA and IgM antigammaglobulins i n rheumatoid arthritis. Arthritis Rheum 14:737-747, 1971
3. Tapanes FJ, Rowson AJ, Hollander JL: Serum antiimmunoglobulins in psoriatic arthritis as compared with
rheumatoid arthritis. Arthritis Rheum 15:153-156, 1972
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patients with rheumatoid arthritis or chronic liver disease
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