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The mechanism of particulate carrier reactions with rheumatoid sera. II. Sensitizing capacity of various human gamma globulins for latex particles

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The Mechanism of Particulate Carrier Reactions with
Rheumatoid Sera. 11. Seiisitizing Capacity of Various
Human Gamma Globulins for Latex Particles
Latex particles can be sensitized by 7s
gamma globulin as well as by aggregated
gamma globulin. The excess of gamma
globulin not on the particle surface may
act as an inhibitor of agglutination if it
is in the aggregated state. Studies are
presented to demonstrate the sensitizing capacity of various human gamma
globulin preparations for latex particles.
Particulas de latex pote esser sensibilisate per globulina gamma 7s e etiam
per globulina gamma aggregate. Le
excess0 de globulina gamma non attachate a1 superficie del particulas pote
ager como inhibitor del agglutination si
ill0 se trova in stato aggregate. Es presentate studios pro demonstrar le capacitate sensibilisatori de varie preparatos
de globulina gamma human pro particulas de latex.
ARTICULATE CARRIERS such as tanned sheep cells,l latex,” collodion3
and bentonite4 particles can adsorb gamma globulin which will react with
certain serum macroglobulins, notably rheumatoid factors (RF), and produce
a characteristic agglutination reaction. Christian“ as well as Edelman et aLg
have demonstrated that the reactant in human gamma globulin (HGG) consists of polydispersed molecular aggregates with a sedimentation constant
greater than 7. These aggregates react with RF in precipitation studies and
are believed to be necessary for sensitization in all serologic systems that
utilize adsorbed HGG.” While it has been shown that aggregates are essential
ior the sensitization of tanned sheep cells, the nature of the sensitizing material
in the latex particle system has not been established.
Fraction 11 materials prepared by the Cohn alcohol fractionation method
are generally used in the F11 L.P. test. The majority of these preparations are
heterogeneous with respect to contaminants from other plasma fractions and
the amount of aggregates. This could conceivably be the reason for the variability of F11 L.P. results in the different laboratories.
It is the purpose of this study to investigate the serologic reactivity of several F11 preparations and to demonstrate that 7s human gamma globulin
(7s HGG) as well as aggregated human gamma globulin (AHGG) can sensitize latex particles.
1. Sixteen Fraction 11 (F11) preparations produced by the Cohn alcohol fractionation
method from pooled human plasma and placental blood were obtained from the following sources: Protein Foundation ( $1014), Hyland ( #HG 67), Pitman-Moore
From the Department of Medicine, State Uniuersity of New York Downstate Medtcal
Center, the Arthritics Clinic and Departments of Medicine and Microbiology, l’he Mount
Sinai Hospital, New York.
Aided in part by grants from the National Institute of Arthritis and Metabolic Diseases,
U . S. P . H . S., and the New York State Chapter of the Arthritis and Rheumatism Foundation.
(#172, ZOO), Cutter ( #1D-2 and B 6085), Lederle ( #C 435, 450, 228A and
Settlings), Squibb (#601 and 103), Amour ( # N 53506), Courtland (#5026,
16076 and 5000A) and Sharp and Dohme ( #30331B). Analysis of these preparations
by paper electrophoresis showed 90-96 per cent gamma globulin. Eight preparations
were analyzed in the ultra-centrifuge and revealed the ususl 7s without evidence of
19s material.' Components sedimenting heavier than 7 s amounted to less than
5 per cent.
2. Two 7s human gamma gloubulin (7s HGG) preparations were used:
a. 7s HGG-a pooled SS, plus SS, fraction, prepared according to the method
of Christian.5
b. 7s HGG II-obtained from the euglobulin fraction of human serum run through
a diethylamino-ethylcellulose column according to the method of Sober et al.7
Analysis by ultracentrifugation showed that this material contained only 7s.
3. Two preparations of aggregated human gamma globulin (AHGG) were used:
a. AHGG &-a pooled SS, plus SS, fraction, prepared according to the method
of Christian.5
b. AHGG II--obtained by further purification of AHGG I which was resuspended
in phosphate buffer ( p H 8 ) and fractionated by sucrose density gradient
( 5-37 per cent) ultracentrifugation. After centrifugation at approximately 80,000
g for three hours in an angle head rotor, the bottom fractions were collected,
dialyzed against buffer and reprecipitated with 2.14 M. sodium sulfate. On
analysis with the ultracentrifuge, the AHGG I1 preparation was found to be
free of nonaggregated 7s material.'
4. Purified rheumatoid factor (RF)," a 1% macroglobulin with a nitrogen contcnt of
1 pg. N/ml. was produced by the method of Heimer et al.8 from the serum of a
patient with rheumatoid arthritis. This preparation was free of 7s material and gave
high titers in the FII latex particle, FII tanned sheep cell, sensitized sheep cell
and sensitized human D erythrocyte tests.
5. Serum #2364 was collected from a patient with rheumatoid arthritis.
6. Latex particles ( L P ) : Dow polystyrene latex particles 0.81 p diameter in a suspension
containing 27.62 per cent so1ids.t
7. Glycine saline buffer: 975 ml. of 0.1 hl glycine and 2.5 nil. of 1 N NaOH were made
up to 1000 ml. with distilled water and the pH was adjusted to 8.2 Ten Gm. of
sodium chloride were then added to each 1000 ml. of buffer.
1. Nibogen contents were measured by the method of Lowry et al.9
2. FII latex particle tests (FII L.P.) were performed according to the method of Singer
and Plotz,z modified by the use of glycine-saline buffer pH 8.2. The stock latex
suspension was prepared by diluting the polystyrene latex particle with distilled
water until 0.1 ml. mixed with 10 ml. of water or buffer matched the light transmission of a standard barium sulfate suspension when examined in a spectrophotometer. The standard barium sulfate solution was made by adding 3 ml. of 0.1 N
barium chloride to 3 ml. of 2,'3 N sulfuric acid and heating at 56 C. for one minute.
Ten ml. of buffer-latex-gamma globulin mixture were prepared by adding 0.1 ml.
of stock latex to 8 ml. glycine-saline buffer, the appropriate amount of gamma globulin,
and then sufficient buffer to make a final volume of 10 ml. This solution contained
4.53~108latex particles per ml. The amount of gamma globulin was expressed as pg.
N/ml. of buffer-latex-gamma globulin solution before mixing with the serum dilutions. An unsensitized latex particle suspension was similarly prepared by omitting
the gamma globulin. Aliquots of 0.5 ml. of buffer-latex-gamma globulin mixture were
added to equal amounts of each seruin dilution and to a control of buffer alone.
After thorough shaking, the tubes were incubated in a water bath at 56 C. for 1%
*Courtesy of Dr. R. Heimer, Hospital for Special Surgery, New York, New York.
{Courtesy of Dr. J. Vanderhoff, Dow Chemical Company, Midland, Michigan.
hours, refrigerated overnight at 4 C. and centrifuged at 2300 r.p.m. for 3 minutes.
The resultant visible agglutination ranging from 1-4+ was read with the naked eye.
Agglutination titers were recorded as the reciprocal of the serum dilution.
Reuctivity of unsensitized LP
Unsensitized latex particles were agglutinated by serum #2346 to a titer of
1600. No agglutination occurred wnen RF was similarly tested. However,
when dilutions of RF were mixed with amounts of 7s IIGG I and 11 ranging
from 1 to 100 pg. N/ml. and unsensitized LP were subsequently added, agglutination occurred in every instance.
Reactivity of LP sensitized FlL
Sixteen FII preparations in concentrations from 0.1 to 500 pg. N/ml. were
used for sensitizing Lk'. in previous experiments these preparauons revealed
a signiiicant degree of variaDility in their capacity to coat tanned sheep cells
and to act as innibitors against the agglutinating activity of KF.'O While some
of the preparations showed strong innibitory activity and were able to sensitize tanned sheep cells in small amounts, others sensitized the cells in lLigh
concentrations only and were weak inhibitors of RF.
In concentrations below 1 pg. N/ml., 10 preparations produced nonspecific
agglutination of LP. At concentrations of 1. to 80 pg. N/ml., three preparations
rendered the LP unstable in the control tubes which made them unsuitable
as sensitizing materials. LP sensitized with the remaining 13 preparations in
concentrations from 10 to 100 ag. Niml. were agglutinated by serum #2364
and RF in titers ranging from 12,800 to 51,200. In this range of sensitization
agglutination endpoints were nearly the same for all preparations. An overail decrease to complete absence of agglutination occurred when LP were
coated with amounts from 125 to 500 pg. N/ml.
Reactivity of LP sensitized with 7s HGG
LP sensitized with 10 to 100 p g . N/ml. of 7s HGG I were agglutinated by
serum #2364 and RF in titers of 12,800 to 25,600. LP sensitized with identical
concentrations of 7s HGG I1 (the purified preparation of 7s HGG I ) gave
similar titers with serum #2364, while with RF the titers were as high as
102,400 (Table 1).When LP were sensitized with 7s HGG I1 previously
heated at 63 C. for 15 minutes and tested with RF, there was a marked fall in
agglutination titers as compared to those for LP sensitized with corresponding
amounts of unheated 7s HGG I1 (Table 2).
Reactivity of LP sensitized with AHGG
When AHGG I and I1 were used as sensitizing agents in corresponding
concentrations, low titers of 1600 were obtained with both serum #2364 and
RF. At coating levels of 150 to 500 pg. N/ml., no agglutination occurred
(Table 1).
Reactivity of LP washed after sensitization
Five ml. aliquots of a stock latex suspension, previously dialyzed against
distilled water, were sensitized with 7 s I-IGG I, FII (Squibb #60l) and
Table 1.-Agglutination Titers of Latex Particles Sensitized with Various
Preparatiosts of liumnn Gamma Globulin*
Serum X2364t
pg. N/ml.
A H G G I1
75 H G G I1
not t a t e d
A H G G I1
not tested
*Prepared from Fraction I1 Squibb 601.
+Titer with unsensitized LP-1,600.
Table 2.-RF
N/ml. added
Agglutination Titers with LP Sensitized with Unheated
and Heated 7s HGG 11
7s H G G 11
Table 3.-Agglutinaiion
heated at 63" for 15 min.
LP sensitized with
Titers of Sensitized LP*
Titers of serum $2364
_ _ - ~ _ _ _ ~ - - _ _ _ ~ - 700 p g . 7s HGG I
700 pg. 7s HGG!63"-60
m h
700 p g . 7s HGG,!63"-60
700 pg. FII Squibb #SO1
700 pg. AHGG I
1400 p g . AHGG I
2800 pg. AHGG I
5600 pg. AHGG I
"Excess of HGG removed by washing.
f Heating of sensitized particles after washing.
AHGG I. Ten minutes after adding the protein, the samples were centrifuged
for 30 minutes at 12,000 r.p.m. The sediments were washed twice, resuspended
in glycine saline buffer and diluted to yield the same light transmission as
latex buffer suspensions in the FII L.P. test. Particles sensitized by this procedure with 700 pg. N of 7s HGG I were agglutinated in the presence of
serum #2364 to a titer of 12,800. Identical titers were obtained when the sensitizing material was heated at 63 C. for 30 or 60 minutes prior to sensitization
and when the LP were washed after sensitization and then heated at 63 C.
for the same time intervals. Particles sensitized with 700 ag. N FII (Squibb
#601) or AHGG I were also agglutinated to a titer of 12,800 by serum
#2364. However, sensitization with 3 to 8 times this amount of AHGG I resulted in a decrease of agglutination titers to 3200 (Table 3). This was probably due to the presence of excess aggregates which could not be removed by
the washing procedure.
In the routine FII L.P. test 0.5 ml. of a 1 per cent solution of FII is added
to 9.5 ml. of latex buffer suspension. This suspension contains 4.53 x lo*
particles per ml., representing a surface area of 9.2 om.* The question arises
as to how much of added protein is actually adsorbed onto the LP surface.
According to the data of Oreskes and Singer,ll this surface area w7ill absorb a
maximum of 1.46 mg. N of HGG. Coating is defined here as the adsorption
of HGG onto the surface of LP, while sensitization may be viewed as coating
which will subsequently react with RF in an agglutination reaction.
At coating levels of 10 to 10 vg. N/ml. there was no significant difference in
the sensitizing capacity of the 13 FII preparations used in this investigation.
These same FII preparations had previously shown mark-ed variability in their
ltbility to sensitize tanned sheep cells in the FII S.C. test.1° When LP were
coated with amounts of FII exceeding 100 pg. N/ml., agglutination titers
dropped sharply or no agglutination occurred at all. It would appear that FII
preparations in amounts from 10 to 100 p g . N/ml. should provide optimum
sensitization and comparable reactivity.
LP sensitized with 10 to 100 pg. N/ml. of 7 s HGG were agglutinated in
high titers, while sensitization with AHGG or heated 7s HGG resulted in
low titers. In experiments where the excess protein was removed with the aid
of high speed centrifugation and washing, LP sensitized by 7s HGG as well as
by AHGG was agglutinated to the same titers and thus both proved to be
effective sensitizing agents. Differences in agglutination titers obtained with
LP not washed after sensitization were presumably due to excess protein
remaining in solution.
The estimated amounts of HGG adsorbed onto particles in the FII L.P.
test at various coating levels are recorded in Table 4. These calculations were
based on an adsorption isotherm, experimentally determined for the binding
of HGG to LP at an alkaline pH.I1 At low concentrations (0.1-1 pgl N/ml.) of
HGG, LP were incompletely coated and frequently unstable in control tubes.
If the particles remained stable, agglutination occurred in relatively low
titers. With larger amounts of HGG for coating, the particle surface approached
saturation and only a small portion of the added HGG was actually utilized;
over 90 per cent of the protein remained free in solution.
AHGG is a strong inhibitor of RF while 7 s HGG shows only weak or no
inhibitory a~tivity.~#6
When AHGG was used as the coating agent in the FII
LP tests, the nonadsorbed material apparently combined with RF, preventing
it, partially or completely, from agglutinating the sensitized particles. If the
coating material contains few aggregates, as is usually the case with FII preparations, inhibitory effect will be less apparent. Resultant titers should be high
except in those instances where the inhibitory effect is enhanced by coating
Table 4.-Amounts
of HGG Adsorbed Onto the Surface of L P in the FZZ L.P. Teat
per cent
HGG added
per cent
adsorbed onto LP
per cent
remaining in solution
of saturation
of LP surface
levels in excess of 100 p g . N/ml. Varying amounts of aggregates in different
FII preparations could nevertheless account for some of the variability in agglutination endpoints with the FII L.P. test encountered in different laboratories. LP coated with 7s HGG were agglutinated in high titers, the excess
protein in solution being the least inhibitory of all HGG preparations.
Purified RF does not react with unsensitized LP. When 7 s HGG was mixed
with R F and unsensitized LP were added, agglutination took place. The use
of a purified RF preparation eliminates the uncontrolled variable of plasma
fractions contained in whole serum. Although no apparent reactions takes
place between RF and 7 s HGG in the FII precipitation and FIT S.C. tests,
R F will react with 7s HGG when it is used as the sensitizing agent in the
latex system. Adsorption or spreading of 7s HGG on the LP surface may
produce a distortion of the reguIarly built molecules of the native protein.
The difference in reactivity of 7 s HGG in the FII S.C. and FII L.F. tests may
be due to a difference in orientation or configuration of the adsorbed molecules
or these two surfaces. The LP, a styrene polymer, has a surface comparatively
less polar than that of the sheep cell. LP sensitized with 7s HGG, washed
and then heated at 63 C., showed no change in serologic reactivity. This suggests that the status of 7s HGG adsorbed onto the surface of LP is not changed
after adsorption.
Tt is postuIated that the adsorption of 7s HGG onto the surface of a LP
leads to the formation of a nucleus carrying muItipIe sites available for
reaction with RF.
Sixteen FII preparations, 7 s human gamma globulin (7s HGG) and aggregated human gamma globulin ( AHGG) were examined for their capacity
to sensitize latex particles (LP) and produce agglutination with rheumatoid
serum and rheumatoid factor (RF) .
In concentrations below 1 pg. N/ml. 10 of 16 FII preparations produced
nonspecific agglutination of LP. Maximum titers were obtained with sensitizing doses of 10 to 100 pg. N/ml. and in this range the endpoint of agglutination
was approximately the same for all Preparations. Titers were significantly lower when FII was used in amounts greater than 100 p g . N/ml. because of excess aagregates in solution.
7s HGG remaining in solution after the coating of LP does not affect agghtination titers whereas heated 7s HGG or AHGG, being reactants for RF,
will markedly reduce the titers.
By removing excess protein after coating. it was demonstrated that 7 s HGG
and AHGG can be aaually effective as sensitizing:materials for latex particles.
The probable mechanism of sensitization in the latex particle system has
been discussed.
The authors are indebted to Dr. R. Heimer of the Hospital for Special Surgery for hi\
invaluable assistance including provision of various gamma globulin preparations and
rheumatoid factor; to Drs. S. S. Schneierson and L. Perlman for their interest and advice;
to Drs. J. W. Ashworth of the American Red Crosq and F. H. Clark of Lederle Laboratories
for the various plasma fractions; and to Dr. Louis Portnoy and Mrs. E. Eason for their
technical assistance.
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ma globulin. J.Exper.Med. 108:105,
M. H. and Kammerer, W. H.: The
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2. Singer, J. M. and Plotz, C. M.: The
latex fixation test. I. Application to
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berg, R. EL: Purification of a rheuarthritis. Am.J.Med. 21.48, 1956.
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3. Wallis, A. D.: Rheumatoid arthritis.
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serolo@cal reaction’
9. Lowry, 0.H., Rosebrough, N. j . , Farr,
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A. L. and Randall, R. T.: Protein
4. Bozicevich, J., Bunim, 1. J., Freund, J.
measurement with the Folin Phenol
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tion test for rheumatoid arthritis.
P ~ ~ ~ . s ~ ~ . EMed.
~ ~ g7:180,
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and Plotz, C. M.: The mechanism of
carrier reactions with
5. Christian, C . L.: Characterization of
111. Adsorption and
the “reactant” (gamma globulin facsensitization of tanned sheep cells by
tor) in the FII precipitin reaction
:md the FII tanned sheep cell aggluvarious human gamma globulin pretinstion test. J.Exper.Med. 708:139,
parations. To be published.
11. Oreskes, I. and Singer, J. M.: The
mechanism of particulate carrier re8. Edelman, G. M., Kunkel, H. G. and
actions. I. Adsorption of human gamFranklin, E. C.: Interaction of the
rheumatoid factor with antigen antima globulin onto polystyrene latex
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body complexes and aggregated gam-
Jacques M. Singer, M.D., Associate Professor of Medicine,
State University of New York, Downstate Medical Center,
Brooklyn, New York; Senior Investigator, Arthritis and Rheumatism Foundation; Research Associate in Medicine, The
Mount Sinai Hospital, New York, New York.
Cideon Altmann, M.S., Head of Department of Bacteriology,
Tel Hashomer Hospital, Israel; Research Fellow in Microbiology, The Mount Sinai Hospital, New York, New York.
Arnold Goldenberg, M.D., Fellow, Arthritis and Rheumatism
Foundation; Research Fellow in Medicine, The Mount Sinai
Hospital, New York, New York.
Charles M. Plotz, M.D., Men. Sc. D., Clinical Assistant Professor of Medicine, State University of New York Downstate
Medical Center, Brooklyn, New York; Chief, Arthritis Clinics,
The Mount Sinai Hospital, New York, and Kings County
Hospital, Brooklyn, New Yark.
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globulin, sera, reaction, capacity, mechanism, latex, human, various, particular, carrier, gamma, sensitizing, particles, rheumatoid
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