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The pH dependence of the sensitized sheep cell reaction of sera from patients with rheumatoid arthritis.

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The pH Dependence of the Seneitbed Sheep Cell Reaction
of Sera from Patients with Rheumatoid arthritis
By RALPHHEIMER,OLGAFEDERICO
AND RICHARDH. FREYBEHG
An effect of pH on the agglutinating
activity of rheumatoid sera was demonstrated in the sensitized sheep cell test
and the potentiated sensitized sheep cell
test. Although the use of a b d e r i n g
system did not change the incidence of
positive tests, it did increase the titer of
some sera. The studies suggest that inhibition of the agglutination system by
some chemicals may be a function of
pH, and that free amino groups may be
essential for agglutination.
Un effect0 de pH super le activitate
agglutinante de seros rheumatoide esseva
demonstrate in le test a sensibilisate
cellulas ovin, simple e potentiate. Ben
que le us0 de un systema de tamponage
non alterava le frequentia de resultatos
positive, ill0 augmentava nonobstante le
titro de certe seros. Le studios suggere
que le inhibition del systema agglutinatori per un substantia chimic es forsan
un function de pH e que libere
amino-gruppos es essential pro le
agglutination.
T
HE SENSITIZED SHEEP CELL REACTION',' and the potentiated
sheep erythrocyte agglutination reaction' have been widely used as a
screening procedure for the diagnosis of rheumatoid arthritis. The basis of
these tests is that serum samples of the majority of patients with active rheumatoid arthritis agglutinate sheep cells sensitized with nonagglutinating concentrations of rabbit (or other species) anti-sheep serum. When the effect
of certain chemicals on the hemagglutination reaction was tested, certain inconsistent results led this laboratory to study the dependence of the titer on
the pH of the agglutinating medium. The effect of certain chemicals believed
to be inhibitors of the hemagglutination reaction was re-evaluated upon
finding the reaction pH dependent. The reasons for the pH dependence of the
hemagglutination titer were investigated, and a mechanism of interaction between the components of the system proposed.
METHODS
Agglutinution procedure. The sensitized sheep cell test was perfonned according to the
method of Heller et a].'; the Potentiated sheep erythrocyte agglutination test was carried
oiit as described by Heller et al." All serum samples were incubated at 56" C for 30
minutes in order to inactivate complement, and heterophilr antibodies were removed by
absorption on equal volames of packed and washed sheep cells.
Use of phosphate bufler in agglutinution procedure. Serum samples tested by the sensitized sheep cell agglutination reaction or by t ) potentiated
~
erythrocyte aggliitination
reaction were serially diluted in isotonic saline or in a four per cent sheep seriirn solution
._
From the Loboruttwy for Research in Rheumlitif: Diseuses, The IIospitd for Special
Surgery, New York 21, New York.
Supported b y a grunt from the Nuticmu1 Institute of Arthritis c i n d Metaholic Diseases,
United States Public Health Service, Bcthesdu, Manjland.
We are indebted to Dr. H . E . Polluczek, Veterans Adrnini,stration Hospital, The Bronx,
New York, for many helpful suggestions.
62
PH
DEPENDENCE OF SHEEP CELL REACTION IN RHEUhlATOID ARTHRITIS
63
respectively, and 0.25 nil. aliquots of each dilution were pipetted in rows along the width
of a 90 place test tube rack. Samples of 0.25 ml. of 0.2 M. phosphate buffer of varying
pH were then added along the length of the test tube rack so that each long row constituted a series of dilutions at a different buffer range. By this procedure pipetting errors
inherent in serial dilution were minimized. A series of phosphate buffers from p H 5.2 to
pH 8 and 0.2 M. KC1-borate buffers above pH 8 were used.
The d e c t of Uiumox, Sulfapyridine and Elkosin on the hemagglutinntion reaction.
To each of 0.25 ml. serially diluted rheumatoid serum, 0.25 ml. of either Diamox
(Lederle; diluted 1:27), Sulfapyridine (Lederle; diluted 1:13) or Elkosin (Ciba; diluted
1 : l O O ) was added. In control experiments, the rheumatoid serum, with and without
added sulfonamides, was serially diluted in phosphate-saline buffer ( p H 7, 0.08 M.).
Treatment of serum samples with iodoacetate, and amino group reagents. Serum samples
wcre diluted fourteen-fold with saline and then dialyzed in the cold for 24 hours against 0.02
M. iodoacetic acid at pH 7. The samples were then redialyzed against excess phosphate
buffer (0.1 M., p H 7 ) . One ml. serum samples were diluted fourteen-fold in bicarbonate
buffer ( 1 M., pH 8 ) and stirred for 3 hours at 4 " C with slow addition of 0.0002 M. of
acetic anhydride or carbobenzoxychloride or fluorodinitrobenzenc. The solutions were
then dialyzed against frequently changed and excess phosphate buffer (0.1 M., pH 7 ) .
The d e c t of acetate and phthalate buffers on the stability of the rheumatoid factor:
Serum samples, diluted in saline, were dialyzed overnight in the cold against 0.1 M.
acetate buffers ( p H range 3.7 to 5.6, in increments of .2 p H units) or against 0.1 M.
phthalate buffers ( p H rangc 2.2 to 3.8 in increments of .2 pH units). The serum samples
wcrc thcn rcdialyzed against excess phospbate-saline buffer ( p H 7, 0.08 M. ).
RESITLTS
The effect of pH on the sensitized sheep cell reaction and the potentiated
sheep erythrocyte agglutination reaction was studied in serum samples of
nine patients with active classical rheumatoid arthritis (table 1 ) . The titers
were pH-dependent in all nine cases. In eight samples the sensitized sheep
cell titers were well below control values when the reaction proceeded at
pH 6.35, dropping to zero at pH 5 2 . The potentiated sheep erythrocyte
agglutination titer of serum s,amples from patients with active classical rheumatoid arthritis also fell off at a similar p€I. Little potentiation of titers by
sheep serum occurred below pH 5.85.
The effect of pH on the titer of serum K from a patient, who six months
subsequent to the testing died of metastatic carcinoma, was particularly interesting. A pH range was found ( p H 7.85 to 8.15) in which this serum sample
had a strongly positive sensitized sheep cell titer (1:448), although at all
other hydrogen ion concentrations the titer remained zero. The potentiated
sheep erythrocyte agglutination titer cjf serum K showed a pronounced
maximum between pH 7.85 and 8.15.
The highest agglutination titers were obtained above pH 7. In some serwn
samples maximal agglutination titers were found over a rather broad range
(sera 104, 102, MM, W, 93 and 98) whereas in some others the optimum
seemed more sharply defined (sera K and U ) . The optimal pH of the sensitized sheep cell reaction seemed to be around pH 8. Borate-saline buffer
( p H 8, 0.1 M.) was included in the sensitized sheep cell tests performed on
a random sampling of test sera, obtained from forty patients treated at the
rheumatic disease clinic, The results were compared with the ones obtained
when no buffer system was used. Table 2 indicates that, whether a buffer
was used or not, the sensitized sheep cell test gave essentially similar results.
5
5%
7%
6
4
7
5
6
8
5
4
8
5%
1
6.20
6.70
PH
ti.90
i.00
i.20
7.50
-___-.7.65
6%
7
7%
7w
8
9
8
8
3
4
4
4
2
3
5
6357
7
7
7
7
5
5
6
5
%
5
5 M
6
6
7%
8
10
10
5%
6
6%
7
6%
8%
9% 8%
9% 10
5 % 7
6 % 8
7
9
7
8%
8%
9% 9%
9%
0
0
0
0
0
4
6
6%
7%
7%
9% 10%
8%
8% 9
12% 12
12
11%
12
4%
4%
5%
6
6%
7
7
7
9
9
8%
8%
10
10
7
9% 10
8
13% 13
10% 13
14
10
5
7%
1
6.36
7
9%
9%
14
11
6
10
8%
11
7
9%
7
6
4
7.X;
11
6
7
12
7
8
3
6%
8.45
11
10
12
10%
12
7
11
11
14%
8
9
8
10
10
5?40
9%
6%
6
7%
4 %
7
6
12
6 x
10
8.15
11
7
13%
6%
9%
0
3
6
6
10
6
8%
h.85
Legend: The numerals in the columns indicate the number of tubes which contained aggliitinnted cells. ‘rhe relationship of the numbers
to the titer of the serriin sample is given by the following:
1
2
3
4
5 etc.
Number:
7 14 28 56 112
Titer 1:
The term “Control” indicates the titer of the serum when tested without addition of hiiffer.
8
12%
ssc
pSEA
7
10
ssc
pSEA
1
0
0
0
5
7
2
2
1
0
0
1
9%
pSEA
13%
0
2
2
2
3
3
2
5
ssc
~~
5
1
4
1
1
4
pSEA
‘
a
2
3
0
3
3
6.05
0
0
1
ssc
10
6
9
6%
~
5.86
___--I.
tlrc IIemogglutiriation Titer Obtuinctl 6~ t l i c Setuitixd Slicep Cell 7 crt (SSC) and the
Potentiuted Sheep Lrytltrcjcyte Test (13-SEA)
6.70
011
0
8%
pSEA
ssc
pSEA
ssc
ssc
1%
1
1%
2
2
1
0
5
9
pSEA
9
pSEA
ssc
pSEA
3
8
6.50
pII
0
1
0
2
6.20
[I\
0
0
0
0
~
Control
l.-’f%c E f f w t
7
ssc
Test
83
h1hf
93
lo4
Serum
No.
?‘AUL.E
2
3
r
4
m
6.5
P H DEPENDENCE OF SHEEP CELL REACTION IN RHEUMATOID ARTHRXTIS
8#)
xQ
7.50
7M
650
6#)l
I
Dilution 1:14
1
128
I
1%
I
I
I
I
I
I
I
I
1412 1:W 1:448 14% 1:1792 13500 1:7T 1:14T
SERIAL DILUTION OF SERUM
FIG.1.-The effect of serial dilution of seriim on the pH of the agglutinating medium.
As can be seen from figure 1, the pH of the agglutinating medium drops
upon dilution of the serum sample. In the sensitized sheep cell test serum
dilutions greater than 1 5 6 are run in pH ranges unfavorable for maximum
agglutination." One would expect higher titers, therefore, in only those sera
which are already positive, whereas serum samples with negative titers
would remain unaffected, as they already fail to agglutinate near optimal
pH conditions. Ten serum samples with titers in excess of 1:swere tested
in the presence as well as absence of borate-saline buffer ( p H 8, 0.1 M . ) .
One to two tube increases in titer occurred in five of the ten samples tested,
while the titer of the other five remained unchanged. Increases in titer beyond two tubes were not encountered. When buffer systems other than
borate were used, results essentially similar to those with phosphate or borate
were obtained. The buffers and buffer ranges were: tris buffer (pH 7.1 to
8.6), barbital (pH 6.8 to 8.2) and glycine (pH 7.6 to 8.4).
TABLE2.-A Summary of the Titers Obtained froin the Sera of Forty Patients Treated at
the Rheumatic Disease Clinic, Using the Sensitized Sheep Cell Test with
and without Borate Saline Bufier
Titers:
NO Buffer
Borate Buffer
(0.1 M., pH 8 )
0
28
29
1:7
1:14
1:28
1:56
1:112
1:234
1:448
2
1
1
1
1
1
1
6
6
0
1
1
0
1
'The potentiated sheep erythrocyte agglutination test affords a more favorable pH
medium for sera of higher titer owing to the buffering capacity of 2 per cent sheep
serum. Some of the potentiation of the agglutination reaction by sheep serum is therefore
due to a shift to a higher and more favorable pH. Sheep serum, however, does contain
a component which i s responsible for potentiated agglutination over and above the pH effect.
For instance, eledrophoretically obtained sheep serum albumin, ox serum iiiucoid fractions,
crystalline lysozyme and the crystalline soy bean trypsin inhibitor failed to potentiate the
sensitized sheep cell reaction.
66
RALPH HEIMER, OLGA FEDERICO AND RICHARD H. FREYBERC.
TABLE3.-The Effect of Stclfonmmidos on ihe Sensitized Sheep Cell Test in Presence or
Absence of p H 7 Phosphate Buffer
Titer
Control
Elkohin
Snlf npyridine
Iliamox
1:448
1:28
1:224
1:s
3.4
8.3
5.9
No bitffer
I
PH
4
c,
E5
Titer
1 :896
1 :896
1 :448
1 :448
PH
7
T
7
7
Phosphate
rn Buffer
TABLE
4.-The Effectof Glycylglycine (0.1 M ) on the Sensitized Sheep Cell Titer of Serum
No. 103 Tested in Presence and Absence of (I Buffer System at p H 7
__-
Titer
Serum No. 103
in
glycylglycine pH 5.4
Serum No. 103
in
glycylglycinc pH 7
adjusted with PO, 1dFt.r
1:224
Serum No. 103
Control
1 :224
_____.__
The pH limitation of the sensitized sheep cell test and the potentiated
sheep erythrocyte agglutination test, however, must be kept in mind when
evaluating the effect of chemicals on the agglutination reaction. On reexamining the alleged inhibitory effect of certain sulionamides on the hemaggliitination reaction4 it was found that direct addition of dilute solutions or
suspensions of commercial sulfonamide preparations ( the preparations were
poorly soluble in saline) to the reacting medium resulted in inhibition of
the hemagglutination test. Decreased titers, however, were directly related
to the pH of the sulfonamide solution added. Upon adjusting the test suspensions to pH 7 no inhibition of the hemagglutination reaction occurred
( table 3 ) . Rheumatoid serum samples, treated directly with sulfonamide
solutions or suspensions and then redialyzed against phosphate buffered saline
( p H 7, 0.08 M.), also had titers identical with untreated controls. These
experiments clearly indicate that “sulfonamide inhibition” is in reality a
pH effect.
Similarly, glycylglycine, reported to be an inhibitor of the sensitized sheep
cell reaction: depressed the hemagglutination titer because of low pH (5.4),
while on adjusting the test suspension to pIf 7 no inhibition occurred (table 4).
A number of other chemicals were tested for their effect on the hemagglutination reaction of sera from patients with rheumatoid arthritis. In all
these experiments care was taken to test for agglutination at a comparable
pH. Table 5 lists some common reagents, the structures affected by them,
and their effect on the aggliitination reaction of rheumatoid sera. These tests
PH
67
DEPENDENCE OF SHEEP CELL REACTION IN RHEUMATOID ARTHRITIS
TABLE5.-Effect of Some Reagents on the Hemagglutination Reaction of Serum Samples
from Individuals with Actbe Classical Rheumatoid Arthritis
REAGENT
GROUP AFFECTED
DEGREE OF INHIBITION
Saturated Periodate
Iodoacwtate
Carbobenzoxychloride
Acetic Anhydride
Fluorodinitrobenzene
Certain Carbohydrates
SH
Amino
Amino
Amino
None
None
++++
+-t++
++++
TABLE&-The pH Requirement for the Irreversible Inactivation of the Rheumatoid Factor
Serum Control
No.
81
101
102
87
122
ioa
83
96
82
U
Titer
1:llZ
1:112
1:112
1:112
1 :224
i : m
1:448
1:448
1:896
1:3600
4.2
1:224
1:112
4.0
1:112
1:66
1:224
1 :896
1 :3600
Sensitized Sheep Cell Agglutination Titers at
pH of acetate or phthalate buffer (0.1 M )
8.8
8.13
8.4
3.2
8.0
2.8
1:112
1:llZ
1:112
l:l4
1:7
0
1:112
1:112
1:112
1:14
0
0
1:66
1:28
1:14
1:14
0
0
1:66
1:66
128
1:14
0
0
1:112
1:112
1:66
1:66
1:14
0
0
1:14
1:14
1:14
1:224
1:66
1:224
1:448
1:896
1:1792
1:448
1:448
1:224
1:1792
1:224
1:448
1:224
1:896
1:112
1:224
1224
1:448
1:66
1:14
1:112
1:224
1:14
0
1:28
1:66
2.6
2.4
0
1:14
0
1:14
1:66
1:14
indicate that the rheumatoid factor does not contain essential SH groups. The
effectof amino group reagents may result either from blocking of the amino
groups or from precipitation.
The stability of the components of the hemagglutination reaction to acid
conditions was also examined (table 6). Rheumatoid serum samples as well
as rabbit anti-sheep serum were dialyzed in the cold against acid buffers,
and then redialyzed against phosphate buffer ( p H 7., 0.08 M.), and the
hydrogen ion concentration determined for irreversible inactivation of the
rheumatoid factor and rabbit anti-sheep serum. In 0.1 M. acetate or phthalate
buffers ( p H 5.0 to pH 2.2) a two tube loss of activity was observed upon exposing the rheumatoid serum samples to between pH 3.8 and 3.2. Mcst of the agglutinating activity of such serum samples disappeared between pH 2.8 and
3.4, depending on the sample tested. Some differences were found in the
pH required for irreversible inactivation, although they were perhaps related
to the initial titer. The decrease in the hemagglutination titer of rheumatoid
sera when the test is carried out below pH 6.3 is, therefore, not due to acid
sensitivity of any of the components of the reaction.
Isoelectric precipitation of one of the participants in the hemagglutination
reaction as a cause for the decreased titer also was considered. Serum samples
and rabbit anti-sheep serum were stored in the cold for 18 hours in phosphate
buffer ( p H 5.85, 0.1 M.) and then centrifuged at 2,000 x g for 20 minutes.
The supernatant was redialyzed against phosphate buffer ( p H 7, 0.08 M.).
The titer of the supernatant remained unchanged, ruling out isoelectric precipitation of any component of the reaction.
DISCUSSION
The pH of the test system is a determining factor for the magnitude of the
68
RALPH HEIMER, OLGA FEDERICO AND RICHARD H. FREYBERG
agglutination between sensitized sheep cells and serum samples from individuals having classical rheumatoid arthritis. Maximal agglutination responses
occur well above pH 7. A pH below 5.8 is unfavorable, while at pH 5.2
complete inhibition of agglutination occurs.
Lacking in buffering capacity, the sensitized sheep cell test in many cases
yields titers which are somewhat lower than those obtained when a proper
buffer is added. Negative titers, however, remain unchanged, as the protein
content of the test samples remains high enough to maintain a nearly optimal
pH for agglutination. The addition of 2 per cent sheep serum as a diluent
of all test samples (the potentiated sheep erythrocyte agglutination test) results in a pH which is more favorable to agglutination. The higher titers
obtained with the potentiated sheep erythrocyte agglutination test are therefore partly a pH effect. However, increases in titer beyond two tubes are
caused by a yet unknown constituent of sheep serum. Such potentiation has
been shown to be a characteristic of serum samples from individuals with
active rheumatoid arthriti~.~
Experimental data presented here suggest that coulombic forces rather
than formation of covalent bonds are involved in this type of agglutination
reaction. Neither isoelectric precipitation nor inactivation by acid of rheumatoid factors and rabbit anti-sheep serum are involved in the inhibition of
agglutination below pH 5.8. Some serum samples, drawn from patients with
collagen diseases other than classic rheumatoid arthritis, do agglutinate sensitized sheep cells even at pH 5.2," which demonstrates that interaction between
rabbit anti-sheep serum and red cell does occur at such a pH. It is therefore
likely that an alteration of electric charge on active sites of the rheumatoid
factors is responsible for such inhibition. The pH range of the inhibition suggests that free amino groups are essential to agglutination, which may only
proceed under relatively basic conditions with the amino group retaining its
free electron pair. Inhibition of agglutination, occurring with weak acid, would
be owing to formation of a center of positive charge on the amino group. The
requirements for agglutination may be schematized as follows:
-NHP:
-NH,+
in acid
does not agglutinate
in base
agglutinates
Blocking of amino groups by acetylation, carbobenzoxylation, and dinitrophenylation, moreover, leads to loss of the ability of rheumatoid sera to
agglutinate sensitized red cells. Whether such inactivation is due to blocking of free amino groups remains to be determined, as such treatment often
leads to formation of insoluble proteins.
SUMMARY
Nine serum samples from individuals with active classical rheumatoid arthritis were examined for the effect of pH on the sensitized sheep cell agglutination titer and the potentiated sheep erythrocyte agglutination titer.
Maximal agglutination occurred above pH 7, while inhibition was noted below
pH 5.8.
PH
DEPENDENCE OF SHEEP CELL REACTION I N RHEUMATOID ARTHRlTIS
69
Routine screening of sera for agglutination activity is scarcely af€ected by
the inclusion of a buffer system. Sera with titers in excess of 156 tend to
exhibit higher titers when a buffer system is used, but negative sera remain
unaffected because the pH of the test suspensions remains at all times near
optimum.
Inhibition of the hemagglutination reaction by addition of certain chemicals
may be a consequence of an altered pH rather than a direct effect exerted by
the chemical. This was shown to be the case for sulfonamides and glycylglycine.
The pH dependence of the hemagglutination reaction is a manifestation of
the electrostatic nature of the reaction.
REFERENCES
1. Rose, H. M., Ragan, C., Pearce, E. and
Lipman, M. 0.: Differential agglutination of normal and sensitized sheep
erythrocytes by sera of patients with
rheumatoid arthritis. Proc. SOC.Exper.
Biol. & Med. 68:1, 1948.
2. Heller, G.,Jacobson, A. S. and Kolodny,
M. H.: A modification of the hemagglutination test for rheumatoid arthritis, Proc. SOC.Erper. Biol. & Med. 72:
316, 1919.
3. -, -, - and Schuman, R. L.: The hemagglutination test for rheumatoid arthritis. I. An immunological analysis of
the factors involved in the reaction.
J. Immunology. 69:27, 1952.
4. Svartz, N. and Schlossmann, K.: Inhibition of the hemagglutination reaction
in rheumatoid arthritis by means of
sulfa compounds. Acta Med. Scand.
155131, 1950.
5. Williams, R. R., Stone, S. S., Jenkins, J.,
Evans, R. L. and Bunim, J. J.: Some
characteristics of the inhibitor system
for the sensitized sheep cell agglutination reaction. Ann. Rheumat. Dis. 15:
61, 1956.
6. Unpublished observations.
Ralph Heimer, Ph.D., Hospital for Specinl Surgery, New York.
Olga Federico, B.A., Hospital for Special Surgery, New York.
Richard H . Freyberg, M.D., Hospital for Specinl Surgery,
New York.
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