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Immunologic studies using antigen-coated charcoalApplication to rheumatoid factor RF.

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Immunologic Studies Using Antigen-Coated Charcoal :
Application to Rheumatoid Factor (RF)
By CHESTERW. GOTTLIEB,KAM-SENG
LAU AND VICTORHERBERT
a number of sensitive
assays for antibodies have been developed using “passive agglutination”.1-2This
consists in the suspension of camer particles in a solution, coating of the carrier
particle with adsorbed antigen, and subsequent visible agglutination of these carrier
particles in the presence of antibody to
their adsorbed antigen. The carrier particles included coll0dion,3-~barium stearate,5 erythrocytes ( especially after treatment with tannic acid) (Boyden),6
b e n t ~ n i t e ,and
~ latex.8 Charcoal particles
had been used to adsorb horse serum
antigen,l schistosomal antigen: and VDRL
antigen,1° but these reports did not identify
the type of charcoal used, nor did they indicate that there is an almost infinite variety
of charcoals. Many charcoals and carbon
blacks are unsuitable to serve as carrier
particles by reason of size, high density,
hydrophobic character requiring an anionic
dispersing agent and an alkaline pH to stay
in aqueous dispersion, non-uniformity, etc.
For these reasons, other workers have been
unable to use charcoal successfully in agglutination reactions.
Our preliminary studies indicated that
charcoals with maximum particle size
greater than 60 microns were not suitable
VER THE YEARS
From the Department of Hematology, The
Mount Sinai Hospital and Medical School, New
York, N e w York 10029
This work was suppwted b y Research Grants
AM 09564 and AM 09062 from the National Institute of Arthritis and Metabolic Diseases of the
United States Public Health Service, and the Albert A. List, Frederick Machlin and Anna Ruth
Lowenberg Funds.
CHESTERW. GOITLIEB, M.D.: Fomwly, Research Assistant, The Department of Hematology.
Current address: captain, M.C., Clinical Investi-
as carrier particles in immunologic reactions. This report presents the results of
studies using a charcoal of smaller than 60micron maximum size. The simplicity of the
protocol here reported suggests its wide
applicability throughout the range of agglutination reactions currently employing
more complex carrier particles. A preliminary report of these findings has been published in abstract form.ll
MATERIALS
AND METHODS
Serum. All sera were stored at -20 C. Serum
from 18 patients with rheumatoid arthritis, supplied by Doctor Irwin Oreskes, had been evaluated
by him for rheumatoid factor ( R F ) using the
latex fixation technic?
Antigen. Lyophilized gamma globulin was purchased from Squibb & Sons, 745 Fifth Avenue,
New York.
Charcoal. Norit A neutral pharmaceutical grade
decolorizing carbon purchased from Amend Drug
and Chemical Co., New York, was used. This
charcoal has a maximum particle size of 53 p
Antigen-coated charcoal. The procedure for
coating charcoal particles is almost instantaneous.
A desired volume of a standard charcoal suspension (generally, 2.5 gm per cent) is mixed with
an equal volume of the desired concentration of
the antigen to be adsorbed in a flask with constant
swirling for one minute. The product is antigencoated charcoal with a ratio of antigen to charcoal
by weight which is dependent on the respective
gations Branch, Medical Research Laboratory,
USA Edgewood Arsenal, Edgewood Arsenal,
Mayland 21010. KAM-SENGLAU, M.D.: Formerly,
Fellow of the World Health Organization at The
Mount Sinai Hospital. Current address: Head,
Clinical Laboratories, Faculty of Medicine, The
University of Malaya, Kuala Lumpur, Malaysia.
VICTORHERBERT,M.D.: Recipient of Career Scientist Award 1-435 from the Health Research
Council of the City of N e w York. Professor of
Medicine, Mount Sinai School of Medicine.
199
A R T H R ~ I AND
S
RHEUMATISM,
VOL. 10, No. 3 (June 1967)
200
GOTTLIEB, LAW AND HERBERT
determined as shown in Table 1. From the
Table it can be seen that uncoated charcoal
sediments rapidly in buffered13 saline and
slowly in the diluted normal and rheumatoid sera (due both to coating of the charcoal by the serum proteins and to retention
of excess serum protein in the suspending
medium). The sedimentation rate is equally
retarded in buffered saline and in dilute
normal human serum with a gamma globulin-to-charcoal ratio by weight between 1:1
and 1:40. When the ratio of gamma globulin to charcoal is reduced to 1:80, the
sedimentation rate in buffered saline is
comparable to the sedimentation rate for
uncoated charcoal; in diluted normal
human serum the sedimentation rate is increased, but is less than uncoated charcoal.
At the 1:80 ratio the gamma globulin coat
is insufficient to retard sedimentation, when
in buffered saline, but may be sufficient to
block additional coating of the charcoal by
the protein in normal human serum. That
normal human serum of itself is insufficient
to retard sedimentation is supported by the
observation that the addition of exogenous
albumin, polyvinylpyrrolidone (PVP ) or
fibrinogen did not further retard the sedimentation of 1:80 gamma globulin-coated
charcoal in normal human serum. Using
low titer ( 1:160) and high titer ( 1:2560)
serum containing RF, sedimentation clearly
more rapid than from normal serum was
evident at gamma globulin-to-charcoal
ratios between 1 : l O and 1:40 (best at
1:40). At a ratio of 1:80, serum containing
low or high titers of RF noticeably inRESULTS
creased the rate of sedimentation compared
One-half ml. of gamma globulin-coated to normal human serum, such that it apNorit A neutral pharmaceutical grade char- proached the rate seen in buffered saline.
Table 2 demonstrates the similar results
coal, which contained 12.5 mg of charcoal
in buffered13 saline to which was added 0.5 of testing 18 sera from patients with rheuml of a 1 : l O dilution of unknown serum, matoid arthritis for RF by the coated charresulted in sedimentation of the charcoal to coal and latex fixation methods. The test
the bottom third of the flocculation tube was performed using a 2.5 gm per cent
within 15 minutes in the presence of RF, suspension of Norit A, coated 1:40 with
but not in the absence of RF. This ratio of gamma globulin (see above). The coated
gamma globulin to charcoal by weight was charcoal results were checked for repro-
concentrations of materials added and the adsorptive capacity of the charcoal for the coating material. When Ilm-labeled albumin was added to
an equal weight of charcoal (Norit A ) , the
charcoal adsorbed approximately 10 per cent of
its weight of the albumin.= Once the coating material has been adsorbed by charcoal it remains
firmly bound, since only a negligible quantity can
be removed by repeated washing.= The charcoal
and coating material were prepared in physiologic
saline or buffered saline. The final suspension was
stored at 5 C. It may be stored for two weeks in
the case of gamma globulin-coated charcoal. Since
the coating process is so simple, it is recommended
that it be carried out on the day of assay.
Sedimentation and Flocculation. Uncoated charcoal particles are hydrophobic and settle rapidly
and completely, forming a pellet at the bottom of
a test tube, with a clear supernatant. Sedimentation is complete within 3 minutes when the concentration of charcoal is 10 to 12.5 mg per ml of
saline diluent (excellent for easy visualization).
Charcoal which has been saturated by the addition
of equal weights of albumin, globulins, fibrinogen,
hemoglobin, Ficoll ( a polymerized alcohol, Pharmacia Laboratories, Inc. ), dextrans, or polyvinylpyrrolidone (PVP),” and then washed and
resuspended in saline, remains dispersed for hours
without significant settling.
Using standard flocculation tubes (10x 75 mm),
0.5 ml of a 1:10 dilution of serum suspected of
containing R F is added to 0.5 ml of a suspension
of 2.5 gm per cent charcoal coated with the appropriate concentration of gamma globulin. The
rate of sedimentation is compared with that of a
control (normal) serum.
The degree of sedimentation was graded from
0 to 4’; 0, no sedimentation; 1+, clearing of the
upper 4s of the coated charcoal suspension; 2+,
clearing of the upper 2/3 of the coated charcoal
suspension; 3+, definite pellet formation at the
bottom of the tube; 4+,clear supernatant with
pellet completely at the bottom of the tube.
1:5
1:10
1 :2u
1 :40
1 :80
3
I)
I,
4f
rr
n
II
"
I,
It
I,
It
n
fI
If
fI
It
I,
I+
n
0
It
I5 min.
4'
huffcrcd saline)
1 :1
1 :2
Time
3 rnin.
Reaction
saline
Buffered
Time
ff
I,
It
I,
tf
4'
w
It
c
It
fl
,I
I#
n
If
n
n
I,
15 min.
rf
4+
W
2*
3+
1+
W
"
0
Time
n
If
I,
n
n
If
I,
10 min.
b
It
I1
,I
If
If
n
15 inin.
Titer 1 :I60
Reaction
~
H
W
fI
PI
4+
3'
14
1'
0
--
I,
5 min.
r,
It
fI
It
"
n
ff
fI
It
10
w
n
n
15 rnin.
Titer 1 :2560
Reaction
Time
-
Serum containing R F
ch arcool ratio for redimentation and flocculation of RF sera
II
f
1+
,
0
Reaction
NHS
of best gamma globulin:
0 :1
(uncoated charcoal)
1 :1
(rcconstituted in
glohutin:
Gamma
-~~~
Table I.-Deberrnination
charcoal
-
*
k
!
!
8
1
2$2
3
202
GOTTLIEB, LAU AND HERBERT
Table 2 . 4 o m p a r i s o n
of
coated charcoal and latex fixation method in 18 sera from patients
with rheumatoid arthritis
Latex
Fixation
Coated Charcoal
(time, minutes)
Reaction
Titer
5
10
15
-
0
0
1'
0
2'
3'
2'
Specimen
Control 396
RA 10210
"
10559
"
10608
"
10609
"
10611
"
10678
"
10750
"
10759
"
10771
"
10802
"
10807
"
10810
"
10838
"
10868
"
10869
"
10891
"
10899
"
10900
____
Neg
Pos
I,
n
n
I,
320
2560
640
320
640
n
5120
I,
2560
5120
n
R
640
n
1280
1280
5120
160
0
,,
N
,I
Neg
R
n
I,
ff
I,
r,
,,
'I
ducibility by making 4 separate runs, each
in duplicate (8 observations for each
sample at each time period). The experimental variation was not greater than 1+
for any serum; the control normal and
RF-negative rheumatoid sera never exceeded I+ (i.e. 1/3 sedimented) at 15
minutes, whereas the RF positive sera were
never less than 2+ (i.e. 2/3 sedimented) at
15 minutes.
The addition of 2 drops of 1:40 gamma
globulin-coated charcoal and 1:10 diluted
serum, respectively, to a slide, resulted in
easily discernible coarsening of the charcoal particles (and clearing of the buffered
saline suspension) in the presence of serum
containing RF but not in its absence, suggesting the usefulness of the procedure as a
rapid slide test. The test was not enhanced
by prior incubation of the reactants for
periods up to one hour, nor by incubation
at 37 C. during the procedure. Buffered
saline was superior to physiological saline
with respect to clarity of the endpoint.
DISCUSSION
Charcoals and carbon blacks are capable
0
0
0
2+
2'
3+
1'
0
0
0
0
3'
0
0
0
0
0
0
'1
1+
3'
3'
4'
2'
3'
1'
'1
2+
3+
1+
0
0
0
1'
0
-
3+
3'
4'
3'
3'
2'
2+
3'
4'
3'
'1
'1
1+
1'
1'
of adsorbing a large variety of protein,
carbohydrate and lipid substances. Particles
of charcoal may, therefore, be coated with
a variety of such substances and should be
sedimented (and flocculated) under appropriate conditions by antibody to these
substances. Thus, in the present communication, charcoal sensitized with gamma
globulin can differentiate, in a rapid sedimentation procedure, sera containing RF
from RF-negative sera. Similarly, hog intrinsic factor (IF)-coated CAL" charcoal
or Carbolac carbon black t in buffered
saline may be sedimented by antibody to
hog IF (made in rabbits).'l
ACKNOWLEDGMENT
We are indebted to Doctor Hugo Pollack, Mrs.
Katherine Kellett and Mr. John Farrelly for technical assistance.
*CAL PULV (5746) charcoal supplied by Basil
Louros, Pittsburgh Activated Carbon Co., Trenton, New Jersey. More than 90 per cent of this
charcoal has a particle size of 44 p or less.
+Supplied by F. A. Heckman, Cabot Corporation, Cambridge, Mass. Carbolac has an 8 to 10
mp particle size and a surface area of approximately 1000 M Z / g m .
IMMUXOLOGIC STUDIES USING ANTIGEN-COATED CHARCOAL
203
SUMMARY
Appropriate charcoals or carbon blacks may be used as carrier particles for antigens
or antibodies in a manner akin to latex, bentonite or erythrocytes. In the present report,
charcoal was substituted for latex in a test for rheumatoid factor. Charcoal and carbon
black may have wide applicability in immunologic sedimentation, flocculation, or agglutination procedures for recognition of antibody or antigen. They are stable, inexpensive, require no preparation or special storage prior to use, and almost instantly
adsorb a wide variety of antigens or antibodies.
SUMMARIOIN INTERLINCUA
Particulas d e varie generes de carbon pote esser usate como vectores de antigeno o de
anticorpore in un maniera simile a latex, bentonita, o erythrocytos. In le studios hic
reportate, carbon esseva usate in loco d e latex in un test pro factor rheumatoide. I1 es
possibile que carbones ha un extense applicabilitate in procedimentos de sedimentation,
flocculation, o agglutination immunologic pro le recognition de anticorpore o antigeno.
Iste materiales es stabile e incostose e require nulle preparation e nulle special mesuras
de preservation ante lor uso. 1110s absorbe quasi instantaneemente un extense varietate
de antigenos o anticorpores.
REFERENCES
1. VeKerdi, L.: Asymmetry of the antigenantibody reaction. Acta Physiol. Acad. Sci.
Hung. 8:91, 1955.
2. Daniel, T. M., and Stavitsky, A. B.: Passive
hemagglutination in study of antigens and
antibodies. In: Methods in Medical Research, Vol. 10, H. Eisen, Ed. Chicago,
Year Book Publishers, 1964, p. 152.
3. Jones, F. S.: Agglutination by precipitin. J.
Exp. Med. 48:183, 1928.
4. Freund, J.: Toxin-antitoxin reaction without
neutralization. J. Exp. Med. 55: 181, 1932.
5. Porter, E. F., and Pappenheimer, A. M.:
Antigen-antibody reactions between layers
adsorbed on built up stearate films. J. Exp.
Med. 69:755, 1939.
6. Boyden, S. V.: The adsorption of proteins on
erythrocytes treated with tannic acid and
subsequent hemagglutination by antiprotein sera. J. Exp. Med. 93:107, 1951.
7 . Bozicevich, J., Tobie, J. E., Thomas, E. H.,
Hoyem, H. M. and Ward, S. B.: A rapid
flocculation test for the diagnosis of trichinosis. Public Health Rep. 66:806, 1951.
8. Singer, J. M. and Plotz, C. M.: The latex
fixation test. I. Application to the serologic
diagnosis of rheumatoid arthritis. Amer. J.
Med. 21:888, 1956.
9. Sadun, E. H., Anderson, R. I., and Schoenbechler, M. J.: A plasma card test for
rapid serodiagnosis of Schistosomiasis
(SPC). Proc. SOC.Exp. Biol. Med. 112:
280, 1963.
10. Portnoy, J., Brewer, J. H., and Harris, A.:
Rapid plasma reagin card test for syphilis and other treponematoses. Public Health
Rep. 77:645, 1962.
11. GoHlieb, c. w., L ~ K.~ s.,, and Herbert,
V.: Immunologic studies using charcoal
particles in sedimentation and flocculation
tests: Application to rheumatoid factor
( R F ) and intrinsic factor (IF). Clin. Res.
13:287, 1965.
12. Gottlieb, C., Lau, K. S., Wasserman, L. R.,
and Herbert, V.: Rapid charcoal assay for
intrinsic factor ( I F ) , gastric juice unsaturated Bls binding capacity, antibody to IF
and serum unsaturated BB binding capacity. Blood 25:875-884, 1965.
13. Herbert, v., Lau, K. s., Gottlieb, c. w. and
Bleicher, S. J.: Coated charcoal immunoassay of insulin. J. Clin. Endocr. 25:13751384, 1965.
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