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Isolation of rheumatoid factors.

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Isolation of Rheumatoid Factors
By RALPHHEIMERAND EDITHR. SCHWAR~Z
'
A method for the separation of rheumatoid factors into two serologically distinct macroglobulins is described.
Macroglobulins, reacting with cells
coated with human as well as with
rabbit gamma globulin, were found to
be mainly 19.4S, but occasionally such
preparations contained minor constituents sedimenting at 22s as well as 27s.
Macroglobulins which reacted with cells
coated with human gamma globulin,
but which were inactive when rabbit
gamma globulin was used, were mainly
19.5s. Some sera, however, contained
substances serologically similar in behavior but sedimenting at 17.5S, 22.OS,
and 27s.
Es describite un methodo pro le separation del factores rheumatoide in duo
serologicamente distincte macroglobuh a s . Esseva constatate que le macroglobulinas que reage con cellulas revestite de globulina gamma human e
etiam con cellulas revestite de globulina
gamma de conilio es principalmente
19,4S,
con-occasionalmente-minor
constituentes que se sedimenta a 22s e
etiam a 27s. Le macroglobulinas que
que reage con cellulas revestite de
globulina gamma human sed que remane
inactive quando globulina gamma de
conilio es usate es principalmente
19,5S. Certe seros contineva substantias
de serologicamente simile comportamento sed sedimentante a 17,5S, 22,0S,
0 27s.
W
HEREAS EARLIER REPORTS'-E of isolation have suggested that the
rheumatoid factor is a single macroglobulin, more recent studies have
shown that the unusual substance in sera of most patients with active classical
rheumatoid arthritis does not meet the criteria of a homogeneous protein.
Heterogeneity, so these studiess-*revealed, was manifest not only on serological
assay but also on physicochemical characterization of the isolated material.
This report* is an account of fractionation of serum samples and purification
of macroglobulins by a combination of absorption techniques, DEAE cellulose
chromatography and sucrose density gradient ultracentrifugation. We aim
to demonstrate that sera of high serological titer contain a number of rheumatoid factors with distinct serological as well as physicochemical characteristics.
MAT~UALS
AND METHODS
Serum Samples.Serum samples, obtained from individuals with classical rheumatoid
arthritis with latex fixation (L.P.F. 11) and sensitized sheep cell (S.S.C.) titers exceeding
1:5120 and 1:8% respectively, were used in this study. Sera were stored in a freezer for
From the Phillp D. Wilson Resenrch Foundaticm and Hospital for Special Surgety, and
Department of Medicine, Cornell Unicersity Medical College, New York, N . Y .
This work was supported in part b y grants from the Nutional Institute of Arthritts and
Metabolic Diseases, Natdolwl Institutes of Health, Bethesda, Md. .
The authors wish to acknowledge the adtiice of R4chard H . Freyberg, M.D., and the
encouragement of their work bg Philip D . Wilson, Sr., M.D. The technical assistance of
Miss Olga M . Federico is also acknotcledged.
"Parts of this report were read at the 134th National Meeting of the American Chemical
Society, Chicago, Ill,, 1958, and the Fifth Interim Scientific Session of the American
Rheumatism Association, Rochestor, Minn.. 1958.
153
154
HEIMER AND SCHWAR'IZ
periods up to six months prior to fractionation. Sixty to 120 ml. of an individual's serum
were used in any single experiment. Control sera obtained from healthy donors with
negative L.P.F. I1 or S.S.C. tests were also used.
Serological Technics.-The L.P.F. I1 test of Singer and Plotz,f' Heller's modification of
the S.S.C. test.'" and the tanned sheep cell test with a human Fraction I1 coating (S.C.F.
II)11 were used. The serological activity of the purified preparations was determined by
serial dilution of one mg./nil. of protein. The last tube showing agglutination was taken
as the end point of the titration.
Protein Analyses.-Serum samples, euglobulin preparations, and purified fractions were
digested by the micro-Kjeldahl technique and analyzed for NH,-t by spectrophotometry
with Nessler's reagent. Where noted, protein concentration was alternately determined
by direct absorption measurements at 278 mp, using a Beckman Spectrophotometer, Model
DU. Optical density was converted to protein concentration by use of a human gamma
globulin ( Squibb, Fraction 11) reference standard.
U l t r u c ~ ~ r i f u g a t i o n . - A ~ ~ l ywere
s e s done in a Spinco Model E ultracentrifuge at 20 C.,
rising cells with double sector centerpieces. The observed sedimentation constants were
corrected to standard conditions and whenever possible corrected to infinite dilution. The
piuified samples were prepared for analysis by dialysis in 0.15M phosphate buffer (pH
5.5). Preparative density gradient ultracentrifugation was performed in a sucrose saline
density gradient as suggested by Fridenberg et al.,*z except that an angle head rotor at
:ipproximately 140,000 x g was used instead of the swinging bucket rotor.
Fructiotuition of the Sera-The
sera were exhaustively absorbed with a thoroughly
washed antigen-antibody complex prepared from sheep erythrocyte stroma and its homologons rabbit antibody. The optimal amount of antibody was determined for each batch of
GJroina. Sufficient quantities of this complex were used for the coinpletv removal of the
sc~uni'sSSC activity. The stroma complex was spun at 4,000 x g in a refrigerated centri
Fiige, washed with ice cold saline, and then treated with 0.15M phosphate buffer ( p H 5.5).
At this pH the rheumatoid factor dissociated from the antigen-antibody complex and was
recovered in the supernatant after centrifugation., To remove considerable quantities of
solub1,e strnnia, the macroglobulin wss further purified by ultracentrifugation in a sucrose
d i n e density gradient. The purified material, henceforth called macroglobulin I, was then
dialyzed in 0.15M phosphate buffer of pH 5.5, and analyzed. Further details of this
purification appear elsewhere.'
The serum fraction, having been absorbed to remove macroglolmlin I, was then
diluted with 10 volumes of distilled water, adjusted to p H 6.1 and kept in the cold for 16
hours. The precipitate which fornied during stcrage was redissolved in 0.15M phosphate
buffer ( p H 7.0) and then dialzed against 0.03M phosphate buffer ( p H 7.0) prior to
fractionation on a DEAE cellulose* column (2.5 x 30 cm). Stepwise elution with buffers
of decreasing pH and increasing ionic strength, being a modification of the procedure of
Lospalluto,a was carried out. The rate of flow was adjusted by a pressure head of nitrogen
to approximately 10 drops per minute. Fractions of 12 ml. each were collected by an
automatic device. Fractions which fixed latex particles in dilutions exceeding 1 : l O were
pooled and concentrated by ultracentrifiigation. T h e pooled fractions were further purified
by density gradient ultracentrifugation in order to remove contaminants sedimenting at
approximately 7s. In this report, these pooled, purified fractions are referred to as macroglobulin 11.
RESULTS
A summary of the macroglobular components of the sera examined and the
rheumatoid factors purified or isolated appears in table 1.
Macroglobulin I preparations were isolated from nine individual serum
samples. The preparations were equally reactive in the SSC, L.P.F. I1 and
*Brown Co., Berlin, N. H.
155
ISOLATION OF RHEUMATOID FACTORS
Table 1.-The Occurrence of Unusual Macroglobulins in Rheumatoid Sera, and the
Zsolution of Rheumtoid Factors from Such Sera'
~~~
Serum
Bj
De
~u
An
St
MacroglobulinI
isolated
Macroglobulin found
in whole serum
Not analyzed
19.4s
(22) 19.4s
(22) z
4s
19.4s
_-
(27) 22,19.4S
Not an&Gi'
23, 19.4s
Ke
( 22 ).4S
(27) (22) 19.4s
27, 22, 1 9 . 4 3 . 5 s
(27) 22, 19.4 (17.5)
We
(22) _
1 9.. 4 r
Le
ul
-_19.4s
- --
S
-~
19.4s
27, iGs
19.4s
19.4s
-
Macroglobulin I1 isolated at
PH 6.6. 0.3 ~r PH6.6, 0.4b p p H 6.2. 0.66 fi
19.5s
-
19.5s
?S
19.4s
None
None
None
(22) 19.5s
?S
?S
19.4s
(22) 19.5s
19.5s
-
(?S)
27,19.5S
22.0s
-
?S
19.5s
_
.
-
--
-
-
None
17.5
~
-
-~
'Major components are underlined; trace components bracketed.
S.C.F.11 tests. The serological reactivity, however, varied from preparation
to preparation. On serial dilution, one mg./ml. of the isolated substances had
titers varying from 1:1O,OOO to 1:60,000.
A macroglobulin with a sedimentation coeflicient of 19.4s was the major
component in all preparations. Six preparations contained no other components.
Minor components sedimenting at 22s (serum De, Pu) or at 27s (serum Ul),
however, were found in three instances. Representative sedimentation patterns
of macroglobulin I preparations appear in figure 1.
Macroglobulin I1 preparations were obtained from each of the seven individual sera selected for such fractionation. Fractions with L.P.F. I1 activity were
found on elution of DEAE cellulose columns with pH 5.5 phosphate buffer
at 0.10M as well as at 0.15M. In three preparations, elution with pH 5.2 phosphate buffer at 0.15M and added 0.1M NaCl yielded an additional fraction
with L.P.F. I1 activity. A representative DEAE cellulose chromatogram (serum
Le) is shown in figure 2. It can be seen that only part of 'the L.P. F. I1 active
material was eluted with 0.10M phosphate buffer of pH 5.5, and that to obtain
the remainder of the serologically active material the eluent's ionic strength
had to be increased.
Elution experiments with sera St and De were carried out in duplicate with
nearly identical results. In experiments with serum U1, the column chromatography was repeated by reapplication of the eluate onto the original column.
The rechromatographed material gave the same elution pattern as the initial
one.
In most chromatographic experiments, nearly all serologically active fractions
were multicomponent systems. Mixtures of 19.5, 22 and 27s components were
encountered, and often up to 40 per cent of the material present was 7s.
Preparations were further purified by a single or multiple, if necessary, sucrose
saline density gradient ultracentrifugation experiments. The progress of one
such purification experiment (serum Le) is shown in figure 3. The starting material was composed of approximately 37 per cent 7S, 58 per cent 19.5S,and 6
per cent 27S, neglecting corrections for the Johnston-Ogsten effect. At the
completion of a density gradient ultracentrifugation run, the contents of the
156
HEIMER AND SCHWARTZ
Fig. 1.-Tracings of sedimentation patterns of purified macroglobulin preparations. ( A ) Macroglobulin I (serum De), 16 minutes after centrifugation at 47,660
RPM, showing a 19.4 and some 22 S component. ( B ) Macroglobulin I (serum Ul),
24 minutes after centrifugation at 47,660 RPM, showing a 19.4 and a 27 S component. ( C ) Macroglobulin 1 (serum An). 16 minutes after Centrifugation at 47,660
RPM. showing a 19.5 S component. ( D ) Illacroglobiilin 11 (serum Ul). 16 minutes
after centrifugation at 47,660 RPM, showing a 22 S component. (E)Macroglobulin
11 (senim St), 24 minutes after centrifugation at 44,770 RPM, showing a 19.5 S
component. ( F ) Macroglobulin I1 (serum U1) , 16 minutes after centrifugation at
44,770 RPM, showing a 17.5 S component.
centrifuge tube were divided into four layers and analyzed for serological activity and distribution of protein components. The two bottom layers, and this
also included the pellet which h d formed during the centrifugation, had the
serological activity with latex particles. The bottom layer (and pellet) (fig. 3C)
consisted mostly of 19.5s and some 27s material, whereas the layer on top of
it (fig. 3 H ) was again mostly 19.5swith a minor component of 7s. The two top
layers (not shown in figure 3) contained 7s material only. Owing to its small
concentration, the 27s component could not be separated from the dominant
19.5s macroglobulin.
By this technique, it was possible in some instances (see table 1) to obtain
preparations containing a single component only.
The sedimentation constants of the isolated macroglobulins were plotted
against the concentration at which these measurements were carried out, and
four such preparations (sera U1, Bj, St and De) had sedimentation coefficients
of approximately 19.5s.Two other preparations (both of serum U1) gave sedimentation coefficients of 17.5s and 22.28 respectively. Tracings of the sedimentation pattern of the 17.5S, 19.5s and 22.2s macroglobulin 11 appear in
figure 1.
ISOLATION OF RHEUMATOID FACTORS
157
Fig. 2.-DEAE cellulose chromatography on serum sample Le following its
absorption by an antigen-antibody complex. One hundred and fifty tubes, containing
12 ml. each were recovered. Latex fixation activity occurred in tubes 105 to 117,
eluted with phosphate buffer of pH 5.5, 0.1 M and in tubes 128 to 139, eluted with
phosphate buffer of pH 5.5, 0.15 M.
Fig. 3.-Progress
of purification of macroglobulin I1 by density gradient
ultracentrifugation. (A) Sedimentation pattern of fraction prior to density gradient
ultracentrifugation. ( B ) Sedimentation pattern of fraction No. 3 obtained after
density gradient ultracentrifugation. (C) Sedimentation pattern of fraction No. 4
obtained after density gradient ultracentrifugation.
As can also be seen in table 1, our isolation techniques yielded on occasion
(sera Bj and Le) macroglobulin I1 preparations, which, although eluting on
158
HEIMER AND SCHWARTZ
chromatography at d i h r e n t points, had identical sedimentation coe5cients.
Because of inadequate concentrations, these macroglobulins could not be compared by electrophoresis, so as to determine whether they are truly different
proteins.
Macroglobulin I1 was uniformly negative in the SSC test. The preparations,
regardless of source of serum or sedimentation coefficient, however, had similar L.P.F. I1 or S.C.F. I1 activities. On serial dilution, the titers of 1 mg./ml. of
macroglobulin 11 ranged from 1:3,000 to 1:6,000. Thus, macroglobuli'n I1 appears to be considerably less potent an agglutinin than macroglobulin I.
DISCUSSION
Evidence is accumulating that the rheumatoid factors, rather than being a
single protein, are a family of closely related macroglobulins. This could already be deduced from the findings of Heller et al.Is and Vaughan.14 The
former demonstrated that sera absorbed with sensitized sheep cells lose all
SSC activity, although they retain nearly unimpaired titers in the S.C.F.I1
test. However, absorption of sera with tanned sheep cells coated with human
gamma globulin causes loss of activity against any and all serological tests.
By use of other antigen-( rabbit) antibody complexes, Vaughan also demonstrated removal of SSC activity from sera without materially affecting S.C.F.
I1 activity. The work reported here shows that by suitable absorption tech.
niques it is possible not only to effect separation of the rheumatoid factor into
two serologically distinct entities, but also to isolate them as distinct proteins.
Oiir work confirms the findings of Lospalluto and Ziff: who were able to
effect separation and isolation of two serologically distinct rheumatoid factors
by DEAE cellulose chromatography.
Macroglobulin I, isolated after its absorption onto and subsequent elution
from the antigen-antibody complex, sheep erythrocyte stroma-rabbit antistroma, differed in its serological behavior from macroglobulin 11. Macroglobulio I was reactive in all known serological tests for rheumatoid factor,
whereas macroglobulin 11, in failing to react with reagents containing rabbit
antibodies as the sensitizing agents, gave positive tests canly with reagents preI1 and L.P.F.I1
pared with human gamma globulin. Furthermore, the S.C.F.
activity of macroglobulin I exceeded that of macroglobulin I1 by a factor of
nearly ten.
Macroglobulin I was shown to sediment mainly at 19.4S,but occasionally
small amounts of 22 and 27s components were also associated with these
fractions. These components could be absorbed by antigen-antibody complexes
and are therefore rheumatoid factors. It is likely that further refinements of
methods for the isolation of macroglobulin I might furnish larger quantities of
22 and 27s macroglobulin. Macroglobulin I1 was shown to occur mainly as a
19.5s material, but depending on the serum chosen for fractionation, a 22s as
well as an unusual 17.5s component were isolated. Although not isolated, a
27s component was also found in one preparation. A 27s macroglobulin was
not found in control studies using sera of healthy subjects.
The behavior of these macroglobulins on DEAE cellulose suggests differ-
159
ISOLATION OF RHEUMATOID FACTORS
ences in electrophoretic mobility. Free electrophoresis of these fractions, however, was not carried out because of lack of material.
Although DEAE cellulose presumably separates the bulk of 7 s gamma globulin from the macrogammaglobulins, the fractions with L.P.F. I1 activity cantained up to 30 per cent of 7s material. The 7 s component was inactive in
the various serological tests for rheumatoid factor, and was eventually removed
by density gradient ultracentrifugation. Chromatographic resolution of macroglobulin I1 was accomplished in duplicate experiments with reproducible
results, and on one occasion the eluted, active fractions were rerun on another
DEAE cellulose column. In this experiment, the serologically active components appeared in the same places as in the original separation. Although the
hazards of stepwise elution of DEAE cellulose columns have been pointed
out repeatedly,16in our hands this method gave better resolution of the different
components of macroglobulin I1 than did gradient elution. Nevertheless,
chromatographic separation of proteins of identical biological activity ( sera
Bj, De, Pu, An, St and Le-see table 1) in the absence of supporting physicochemical data cannot be taken as absolute proof of their heterogeneous nature.
Although the variability of rheumatoid factors from individual to individual
has not been established so far, the results of our investigations, as well as
those of Kunkel and his collaborators,lG suggest individual variability. These
studies, done on concentrates of sera, demonstrate differences in the macroglobular profile of sera of individuals with rheumatoid arthritis. Demonstrable
differences in the ultracentrifuge. however, can be shown with sera of high
titer only, and therefore comparatively few sera are amenable to this analysis.
It can be seen from table 1that a selected group of sera had a varying content
of 17.5, 19, 22, and 27s macroglobulin. The isolation of 17.5S, 22s and 27s
rheumatoid factors, therefore, is dependent on the choice of the starting material.
Control sera of healthy subjects failed to yield macroglobulins with the
serological activity of the rheumatoid factor. However, macroglobulins other
than rheumatoid factor were obtained on DEAE cellulose chromatography of
control sera. These macroglobulins, sedimenting at about 19.5S, but never
at 22 or 27S, are likely contaminants of our macroglobulin I1 preparations.
SUMMARY
A method for the separation of rheumatoid factors into two serologically
distinct macroglobulins is described. Macroglobulins, reacting with cells
coated with human as well as with rabbit gamma globulin, were found to be
mainly 19.4S, but occasionally such preparations contained minor constituents
sedimenting at 22s as well as 27s. Macroglobulins which reacted with cells
coated with human gamma globulin, but which were inactive when rabbit
gamma globulin was used, were mainly 19.5s. Some sera, however, contained
substances serologically similar in behavior but sedimenting at 17.5S, 22.OS,
and 27s.
REFERENCES
1. Kunkel, H. G., Franklin, E. C., and
Muller-Eberhard, H. J.: Studies on
the isolation and characterization of
the “rheumatoid factor.” J. Clin.
In-
160
HEIMER AND SCHWAR'IZ
ritis. Proc. SOC. Exper. Biol. & Med.
vest. 38:424, 1959.
72:316, 1949.
2. Lospalluto, J., Lewis, A. and Ziff, hl.:
Isolation of the rheumatoid factor 11. -., Jacobson, A. S., Kolodny, M. H.,
and Kammerer, \Y. €1.: The heniag(Abstract), J. Clin. Invest. 37:913,
glutination test for rheumatoid ar1858.
thritis. 11. The influence of human
3. svark, N., Carlson, L. A., Schlossplasma fraction I1 on the reaction. J.
mann, K., and Ehrenberg, .4.: IsolaImmunol. 72:66, 1954.
tion of the rheumatoid factor. Acta
12. Fudenberg, H. H. and Kunkel, H. G.:
med. scandinav. 160r87, 1958.
Physical properties of red cell ag4. Heimer, R., Federico, 0.M., and Freyglutinins in acquired hemolytic aneberg, R. H.: Purification of a rheumamia. J. Exper. hled. 106:689, 1957.
toid factor. Proc. Soc. Exper. Biol. &
13. Heller, G., Kolodny, hl. H., Lepow,
Med. 99:381, 1958.
I. H., Jacobson, A. S., Rivera, M. E.,
5. Williams, R. R., Stewart, L. C., and
and Marks, G. H.: The hemagglutinaJenkins, J. C.: Purification and isolation test for rheumatoid arthritis. IV.
tion of rheumatoid factor. Proc. SOC.
Characterization of rheumatoid agExper. Biol. & Med. 99:554, 1958.
glutinating factors by analysis of se6. Lospalluto, J,, and Ziff, M.: Chromatorum fractions prepared by ethanol
graphic studies on the rheumatoid
fractionation. J. Immunol. 74340,
factor. J. Exper. hled. Il0:169, 1959.
1955.
7. Heimer, R., Schwartz, E. R., and Freyberg, R. H.: Presented at the sixth 14. Vaughan, J. H.: Behavior of the rheumatoid arthritis agglutinating factor
Interim Session of the American
with immune precipitates. J. ImRheumatism Association, Detroit,
munol. 77:181, 1956.
Mich., December 1959.
8. Franklin, E. C.: The precipitin reaction 15. Sober, A., and Peterson, E. A.: Chromatographic evnluation of protein
between rheumatoid factors and gammixtures in Amino Acids, Proteins
ma globulin: Studies by double difand Cancer Biochemistry. New York,
fusion in agar. Arth. ti Rheumat. 3:
Academic Press, 1960.
16, 1960.
9. Singer, J. M. and Plotz, C. M.: The 16. Franklin, E. C.,Holman, H. R., MullerEberhard, H. J., and Kunkel, H. G.:
latex fixation test, I. Application to
An unusual protein component of
the serologic diagnosis of rheumatoid
high molecular weight in the s e m
arthritis. Am. J. Med. 21:888, 1956.
of certain patients with rheumatoid
10. Heller, G.,Jacobson, A. S., and Kolodny,
arthritis. J. Exper. Med. 105425,
M. H.: A modification of the hemag1957.
glutination test for rheumatoid arth-
Ralph Heimer, Ph.D., Research Associate, Hospital for SpecMl
Surgery; Research Associate, Department of Medicine, Cornell
University Medical College, New York,N . Y .
Edith R. Schwartz, B.S., A.M., Research Assistant, Hospital
for Special Surgery, New York, N . Y.
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