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Journal of Experimental Medicine

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BARIUM SULPHATE ABSORPTION AND THE
DIAGNOSIS OF SYPHILIS.*SERUM

BY HIDEYO NOGUCHI ,AND J. BRONFENBRENNER.
(From the Laboratories of the Rockefeller Institute for Medical Research,
New York.)

Negative serum reactions in untreated, well-developed cases ofsecondary syphilis have been sometimes encountered by those who
employ the original method of Wa'ssermann. According to Wechselmann, whose experiments were carried out under Wassermann,
this is said to be due to the development of complementoid in thepatient's serum during inactivation, and inactivation is essential for
Wassermann's method. Wechelsmann states that the treatment ofsuch specimens of syphilitic sera with barium sulphate removes theinterfering complementoid and causes the treated sera to react positively. A later report by Lange contains a list of a large numberof sera which either became positive or stronger after treatment
with barium sulphate. According to Wechselmann and Lange,
every specimen of serum sent for diagnosis must be treated withbarium sulphate in order to insure a reliable result. Although theabsorption of serum with barium sulphate is a simple enough process, yet before its routine adoption in the complement fixation testby the Wassermann method, further examination is demanded.
Whatever may be the cause, the phenomenon itself is of interest.
In our studies of this subject we employed the antihuman hemolytic
system, and have considered the following questions:I. The antibody content before and after barium sulphate absorption.
2. The effect of barium sulphate upon the complementary activityof guinea pig serum.
3- The effect of barium sulphate upon the antibody content ofdifferent syphilitic sera.
* Received for publication, September 2I, I9IO.
217

218

Barium Sulphate and the Serum Diagnosis of Syphilis.

4. The effect of the addition of normal serum upon the barium
sulphate absorption of syphilitic antibody.
5- The fate of syphilitic antibodies after barium sulphate absorption.
6. The absorption of hemolytic amboceptors by barium sulphate.

I. The Antibody Content before and after Barium Sulphate
Absorption.--In this series of experiments we employed unheated
and inactivated syphilitic sera. The mixtures of the serum andbarium sulphate were made in the proportions originally recommended by Wechselmann. The result shows that the absorption of
unheated as well as inactivated serum renders the reaction somewhat stronger, especially with the inactivated sera. This experiment confirms the finding of Wechselmann with inactivated serum,
but does not prove that it was the removal of the complementoidthat made the reaction stronger. The barium sulphate removed
certain serum constituents which interfere with the fixation of thecomplement. The increase in the intensity of reaction after the
T A B L E I.
Absorption with BaS04 Increases the Number of Antibody Units.
U n h e a t e d syphilitic serum.

Syphilitic serumo.x c.c. + B a S O 4 (7 56)

0.055 c.c.; incubation ;centrifugalization.

The centrlfugate wasmade equal to 5 c.c.
I with salt solution andused for the fixation test. I

T h e s a m e s e r u m after i n a c t i v a t i o n .

Syphilitic serum( 5 6 o c . ) o.z c.c.

+ BaSO4 (79)o, o55 c.c.; i n c u b a t i o n ;C o n t r o l without B a S O 4
centrifugalization.
treatment.

Control without BaSO4
treatment.

T h e centrifugate wasm a d e e q u a l to 5 c.c.
w i t h salt solution a n d
u s e d for the fixation test.

No hemolysis
No hemolysis
No hemolysis
No hemolysis
No hemolysis
No hemolysis
No hemolysis
No hemolysis
Slight hemolysis
No hemolysis
No hemolysis
No hemolysis
M u c h hemolysis
No hemolysis
No hemolysis
No hemolysis
Complete hemolysis
M u c h hemolysis
No hemolysis
No hemolysis
Complete hemolysis Complete hemolysis
No hemolysis
No hemolysis
Complete hemolysis Complete hemolysis
No hemolysis
No hemolysis
Complete hemolysis Complete hemolysis
No hemolysis
No hemolysis~ ' ~ 0.2
Complete hemolysis Complete hemolysis
Trace hemolysis
Slight hemolysis
Moderate hemolysis Complete hemolysis Complete hemolysis Complete hemolysis~ ~ ¢9 O . I~ ~ 0.075 Complete hemolysis Complete hemolysis Complete hemolysis Complete hemolysis
Complete _hemolysis] Complete hemolysis Complete hemolysis Complete hemolysis<2o.o5
5 =6.6
5--- =IO
=30
5_
=2s.s
0.75
0.5
o.io
o.I75
antibody units
antibody units
antibody units] antibody units
x.o
0.75
0.625
o.s
"CvO ~~ ~9 0.4,.~ ~ -~ o.3*~ ~= ~ 0.25
on2

219

Hideyo Noguchi and J. Bronfenbrenner.

barium sulphate absorption is almost negligible with the unheatedsera.
The results in table I show that a certain quantity of barium
sulphate mixed with the syphilitic serum strengthens the reaction.
It was of interest, therefore, to see whether barium sulphate inother proportions might not remove the syphilitic antibody. The
results of experiments determining this point are recorded in table
II.
TABLE II.

Removal of Syphilitic Antibody by Absorption with BaS04.
Syphilitic serum No. ~.
0.002 C.C.

BaSO4 (7 per cent.)
I.o
0.7
0.5
0.4
o.3
0.25
0.2

o.I5
O.I

0.07
O.05
O.O4
O.O3
0.025
0.02

o.oi5
0.01

0.007
o.oo5
control
Amount of BaSO4 (7 percent.) required forcomplete absorption.

Syphilitic serum No. 2.
o.~3c.c.

o.o3c.c.

Complete H.
Complete H.
Complete H.
Complete H.
Complete It.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Much H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.

Complete H.
Complete H.
Complete H.
Complete H.
Much H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.

Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Much H.
No H.
No H.
No H.
No H.
No H.

Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Much H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.
No H.

0.04 c.c.

0.4 c.c.

0.025 e.c.

0.3 c . c .

H = hemolysis.

Table II shows that the syphilitic antibody can be completely
removed by absorption with a sufficiently large amount of barium
sulphate. With a given serum the amounts of the antibodies removed by barium sulphate are approximately proportional to theamounts of barium sulphate employed. Thus, to remove ten times
the number of antibody units, ten times the quantity of barium
sulphate is required. On the other hand, we have noticed that the220

Barium Sulphate and the Serum Diagnosis of Syphilis.

absolute amount of the sera containing the antibodies bears noquantitative relationship to the amount of barium sulphate requiredfor different sera. In the experiment (table I I ) , a somewhat
larger amount of barium sulphate is seen to be necessary to remove
the antibodies from a somewhat smaller amount of the serum. The
number of antibody units contained in 0.002 cubic centimeter of thefirst specimen was the same as that in 0.003 cubic centimeter of thesecond. From this fact it is clear that the absorption by barium
sulphate is not directed in a selective manner to the antibodies.

2. The Effect of Barium Sulphate upon the Complementary Activity of Guinea Pig Serum.--Incidentally, it may be mentionedthat barium sulphate has almost no anticomplementary action uponguinea pig serum. A slight antihemolytic effect has been noticed
when one cubic centimeter of a 7 per cent. suspension of this salt
was mixed with 0.05 cubic centimeter of the complement.

3. The Effect of Barium Sulphate upon the Antibody Content
of Different Syphilitic Sera.--In this series of experiments weselected six samples of syphilitic sera, the antibody contents ofwhich were very variable. The titers of these sera are recorded
in table III, where other details of the experiments are also given.
The results were rather unexpected and reveal wide variationsin the ease or difficulty with which the removal of the antibodies
from different sera could be effected with barium sulphate absorption. At the foot of the table we have recorded figures for theamounts of barium sulphate necessary for removing one antibody
unit from different sera and the relative quantities of the barium
salt necessary to absorb a given unit volume of these six sera. This
numerical expression aids us in seeing how each specimen behaved
towards the barium sulphate absorption. The most difficult serum
to fix, No. I, is found to be nearly forty times less sensitive to thebarium sulphate absorption than the most' easily fixable specimenof serum, No. 2. Here again we obtain the evidence that theabsorption by barium sulphate is not proportional to the amount of
the serum. Nor is it parallel to the antibody unit, because theamount of each serum employed in this experiment represented
about one unit. If the absorption were selectively directed toward
the antibodies, the amount of barium sulphate necessary to remove

221

Hideyo Noguchi and J. Bronfenbrenner.
T A B L E III.

Antibody Content of Different Sera Removed by BaS04.
Determination of the quantities of BaSO 4 necessary to remove x unit of syphilitic antibody fromdifferent samples of positive sera.

BaSO4(7 ~ )
0.5
0.4
0.3
0.2
0.1

Serum No. x.
Titer,

Serum No. 2.
Titer,

Serum No. 3.
Titer,

Serum No. 4Titer,

Serum No. 5.
Titer,

Serum No. 6.
Titer,

0.00125 C . C .

0.00175 c.e.

0.0025 c.c.

o.oi25 c.c.

o.oi25 c.c.

0.025 c.c.

Complete H.
Complete H.
Complete H.
Complete H.
No H.

No H.

Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
Complete H.
No H.
No H.

0 . 2 C.C.

0 . 0 0 7 C.C.

0.07
0.05
o.o4
o.o3
0.02

o.o15
O.OI

0.007
0.005
control
BaSO4 for removal of unit
antibody

BaSO4 for re0.2
moval of I
= I60
volume unit 0 . o o i 2 5of serum

0.007

0.o017---~ = 4

Complete H. Complete H. Complete H.
Complete H. Complete H. Complete H.
Complete H. Complete H. Complete H.
Complete H.
Complete H.
No H.
Moderate H.
Complete H.
No H.
N o H.

[I

Complete H.
Complete H.
Complete H.
Complete H.
No H.

No H.

No H.

No H.

No H.

0.1 c.e.

0.3 c.c.

0 . 2 C.C.

0.2C.C.

0.3

0.I

0.002540

O.01250.2
24

o.o125

= I6

0.2

0.025

=8

I-I = h e m o l y s i s .

the antibodies would have been nearly the same for all specimens.
Just what determines this variation in the fixability of humanserum by barium sulphate was not investigated further.
In the next series of experiments we determined whether barium
sulphate absorbs the anticomplementary substances of human serum
as in the case of the syphilitic antibodies. For this purpose we
selected some syphilitic sera which became anticomplementary upon
standing for many days. The results are recorded in table IV.
The experiments in table IV demonstrate that barium sulphateremoves both the anticomplementary substances and the syphilitic
antibodies.

4. The Effect of the Addition of Normal Serum Upon the Barium
Sulphate Absorption of Syphilitic Antibody.--We have alreadyshown that barium sulphate removes the antibodies from a syphilitic

Barium Sulphate and the Serum Diagnosis of Syphilis.

222

0

0

i¸

==

o

r.r.l

o

"
~

~

.

.~-

~

4o

~

C~

r~

0

o

0

~

~ ~.~

6~6
0

°?

0

0

Z

66

~

6~6

6~6
0

B
B
o
o

0

0

~6~6
0

0

Hideyo Noguchi and J. Bronfenbrenner.

223

serum in almost direct proportion to the amount of this salt used
for absorption. W e have also stated that the treatment of somesyphilitic sera with a comparatively small amount of barium sulphate removes certain serum constituents and renders the reaction
somewhat stronger than in the untreated serum. W e have also
shown elsewhere that when the syphilitic antibody is associated withcomparatively large amounts of normal serum constituents, thefixation reaction is proportionately less sensitive. Reversing thisconsideration, we derive the conclusion that by removing the normal serum constituents as much as possible we can make the reaction more distinct; in other words, we might be able to purify
the syphilitic antibodies. Such a purification is possible, especiallywhen the other interfering serum constituents possess a greateraffinity than the antibodies for barium sulphate. It is also possible
to purify the syphilitic antibodies to a certain extent, even when
the affinities of the antibodies and other serum constituents for barium sulphate absorption are the same, provided the amount of theserum constituents is greater than that of the antibodies. This is
not possible, however, when the amounts of the indifferent serum
constituents and the antibodies are in the reverse proportion, because here we would remove more antibodies than interfering
serum constituents. It is, therefore, necessary to determine experimentally whether the non-syphilitic serum possesses the sameaffinity for barium sulphate as the antibodies of syphilitic serum
do. W e have accordingly estimated the number of the antibody
units after barium sulphate absorption in syphilitic serum, syphilitic serum plus an equal amount of normal serum, and syphilitic
serum plus four times the quantity of normal serum. If thenormal serum possesses the same affinity as the syphilitic antibodies,
its presence must interfere with the removal of the latter by a given
amount of barium sulphate.
The protocol given in table V shows that the addition of one
part of normal serum to one part of syphilitic serum reduced theremoval of the antibodies to one-half of that obtained when no normal serum was added, and the addition of four parts of the normal
serum to one part of syphilitic serum reduced the removal to about
one-fifth. This indicated undoubtedly that the normal serum e m -224

Barium Sulphate and the Serum Diagnosis of Syphilis.
TABLE

V.

Interference by Normal Serum with the BaS04 Absorption of Syphilitic Antibody.
Series i.
Syphilitic serum and
BaSO4.
Amounts.of
diluted centrifugate
Syphilitic serum
used in the
fixation test (x:Io) x.2 c.c. + B a S O 4( 7 5 ) o.5 c . c . ; i n c u b a witht i o n a t 37 ° C., x hour ;antigen.
centrifugalization.

The centrlfugate ismade equal to 25 c.c.
with salt solution.
i.o
0.7
0.5
0.4
0.3
0.25
0.2
O.X5
O.I
0.07
0.05
o.04
o.o3

Series 2.

Series 3.

Series 4-

Syphilitic serum andnormal serum, equalparts, and BaS04.

Syphilitic serum with
4 parts of normal sermu, and BaSO 4.

Control : syphilitic
serum alone without
BaSO 4.

Syphilitic serum( z : m ) 1.2 c , c . + n o r mal serum (I:m)
x.2 c . c . + B a S O 4 ( 7 ~ )0.6 c c.; i n c u b a t i o n ;centrifugalization.

Syphilitic serum(x:xo) x.~ c.c. + n o r mal serum lundiluted
0. 5 c.c. + BaSO 4 (7~o.6 c . c . ; i n c u b a t i o nuentrifugalization.

Syphilitic serum(x:xo) x.2 c . c . + s a l t
s o l u t i o n 0.6 c . c . ; i n cubation i centrifugalization.

The centrifugate ismade equal to 25 c.c.
with salt solution.

The centrifugate ismade equal to 25 c.c.
with salt solution.

The fluid is madeequal to 25 c.c. withsalt solution.

No hemolysis.
N o hemolysis.
N o hemolysis.
N o hemolysis.
N o hemolysis•
N o hemolysis.
N o hemolysis.
N o hemolysis.
N o hemolysis.
N o hemolysis.
N o hemolysis.
N o hemolysis.
AlmostN o hemolysis•
N o hemolysis.
C o m p l e t e hemolysis.!i c o m p l e t e hemolysis.
No hemolysis.
C o m p l e t e hemolysis. C o m p l e t e hemolysis.I T r a c e h e m o l y s i s .
C o m p l e t e hemolysis. C o m p l e t e hemolysis. M u c h hemolysis,
C o m p l e t e hemolysis. C o m p l e t e hemolysis. C o m p l e t e hemolysis.
C o m p l e t e hemolysis. C o m p l e t e hemolysis. C o m p l e t e hemolysis.
C o m p l e t e hemolysis. C o m p l e t e hemolysis.
C o m p l e t e hemolysis.
C o m p l e t e hemolysis.
C o m p l e t e hemolysis.

N o hemolysis.
M u c h hemolysis.
C o m p l e t e hemolysis.
C o m p l e t e hemolysis.

=25

units;

r e m o v e d 75 u n i t s ;O•4=62.5
units;•

r e m o v e d 37.5 u n i t s ;25 =83.3 units;o•3
r e m o v e d I6.6 u n i t s ;255 = i o o u n i t s ;0.25
original content.

ployed, possessed the same affinity as the syphilitic antibodies forbarium sulphate. In another experiment we found that the numberof demonstrable antibodies in a mixture of normal and syphilitic
sera, in the proportions of I to I and I to 4, was considerably
smaller when barium sulphate was not used than when it was used.
As we have shown in another communication, when normal serum s
is added to a mixture containing syphilitic antigen and antibody, itinterferes with the fixation of complement subsequently to be added,by uniting with the molecules which under the usual conditions arecapable of fixing complement.
It would manifestly be unsafe to conclude from these experiments that all normal sera behave in the same way. On the con1 In fixation experiments, white of egg has a strong interfering property which
can be removed to a large extent by absorption with barium sulphate.

225

Hideyo Noguchi and J. Bronfenbrenner.

trary, we already know that there are many human sera which fix
with great difficulty.

5. The Fate of Syphilitic Antibodies after Barium Sulphate Absorption.--We have endeavored to trace the antibodies which disappear during the process of barium sulphate absorption.
results are given in table VI.
TABLE

The

VI.

Fate of the Syphilitic Antibody after BaSO, Absorption.
Syphilitic s e r u m {x:2o) 0.6 c.c.

+ BASO4(756)
o.6 c.c.;incubation ;centrlfugalization.

Control : no BaS04
absorption ; syphilitic s e r u m (x:2o)
C
e
n
t
r
i
f
u
g
a
t
e
w
a
s
Sediment resuspendo.6
c.c. i n 4 c.c. salt
m a d e e q u a l to 4 c.c.
e d i n 4 c.c. salt
solution.
w
i
t
h
salt
s
o
l
u
t
i
o
n
solution, and used
a n d u s e d for f i x a t i o n
for fixation test.
test.

Q u a n t i t i e s of: (I) s e d i m e n tsuspension,
(2)
BaSO~
t r e a t e d serum, (3) unt r e a t e d s e r u m (control)
for fixation test. C o m p l e m e n t , o.o5 c.c. T h e r e s t
of t h e p r o c e d u r e w a s as
usual.

1.0
0.8
o.6
o.5
0.4
0.3
0.25
0.2
0.I

N u m b e r of a n t i b o d y u n i t s cont a i n e d in t h e t o t a l 4 c.c. of eachseries.

N o H.
N o H.
N o H.
C o m p l e t e H.
C o m p l e t e H.
Complete H.
Complete H.
IO units.

N o H.
M o d e r a t e H.
C o m p l e t e H.
C o m p l e t e H.
C o m p l e t e H.

4 units.

N o H.
N o H.
N o H.
N o H.
N o H.
M u c h H.
C o m p l e t e H.
I6 u n i t s .

The experiments given in table VI show ( I ) that the syphilitic
antibodies are removed by barium sulphate (to which they seem toadhere), and (2) that the antibodies thus absorbed retain theiractivity.

6. The Ab.¢orption of Hemolytic Amboceptors by Barimn Sulphate.--Several experiments were performed with antihumanamboceptor (rabbit), and the results show that the amboceptpor isquite readily removed from the solution by barium sulphate absorption. The barium sulphate treated with the amboceptor solution
retains the amboceptor and causes hemolysis when mixed withhuman corpuscles and guinea pig complement. Although we havenot made similar absorption tests with antisheep amboceptor, it isprobal~le that this acts in the same way.

226

Barium Sulphate and the Serum Diagnosis of Syphilis.
CONCLUSIONS.

The so-called syphilitic antibodies can be removed from a serumby means of absorption with barium sulphate. The removal is due
either to an adsorption or a mechanical absorption. The activityof the syphilitic antibodies is thereby unimpaired. The readinesswith which the absorption is accomplished with barium sulphatevaries considerably with different syphilitic sera. That barium
sulphate exerts the same absorbing effect upon non-syphilitic serum
components is made evident by the interfering property which thelatter manifest in the absorption experiment of the syphilitic antibodies. The selective removal of the serum components, otherthan the syphilitic antibodies, by means of barium sulphate absorption is, therefore, impossible.
On the other hand, a partial removal of these components, withbut little removal of the syphilitic antibodies, may be effected when
the content of a given serum is poor in syphilitic antibodies andcomparatively rich in the indifferent serum components. But thisis impossible if the conditions are reversed. The main reasons
why some negative syphilitic sera may be so modified by thebarium sulphate treatment as to give positive reactions, areexplained below, but these apply only to those methods inwhich inactivated serum is employed. The inactivation reduces
the antibody content to about one-fourth to one-fifth of theoriginal. When the serum is very rich in antibodies, this does notaffect the result of the fixation test. But when the amount of theantibodies is small, the process of inactivation creates conditionsquite unexpected. It may produce such a condition that a given
amount of the serum contains, after inactivation, only one or two
antibody units, while the other serum components remain undiminished. Here one must not lose sight of the vital fact thatthese apparently indifferent serum constituents are not at all indifferent in the fixation processes. They may possess affinities
which are similar to those of complement for the fixing combination of syphilitic serum and antigen. Speaking quantitatively, one
unit of the syphilitic antibodies plus antigen will fix o.I cubic centimeter of guinea pig complement, but this unit can also be saturated
and blocked by nearly the same amount of the seemingly indifferent

Hideyo Noguehi and J. Bronfenbrenner.

227

serum component of the serum to be tested. Moreover, the regularamount of inactivated serum used in the Wassermann system is o.2
cubic centimeter, a quantity sufficient to saturate two units of the
fixing combination. Fortunately, this self-saturation of the syphilitic antibody-antigen combination by the other serum components
is not constant in occurrence, owing to the wide variations of the
fixability of the serum components of man. Here the benefit ofWechselmann's procedure becomes obvious. By removing a surplus of the fixable indifferent serum components by means of barium
sulphate, the serum is made to react positively, or more strongly
than before the treatment with barium sulphate. This masking of
the positive reaction through the self-saturation is liable to occur
in any s3~stem in which inactivated serum is recommended.
Another equally important factor in masking the positive reaction
in a serum in which the antibodies are poor, is the presence in considerable amount of natural antisheep amboceptor in human serum.
It is a plain and simple fact that an excess of hemolytic amboceptor
renders a positive reaction feeble or completely negative. As wehave shown in our present investigation, a hemolytic amboceptor
can be removed from the serum by means of absorption with barium
sulphate. Thus it is easy to understand why Wechselmann foundthat barium sulphate absorption improves the reaction in theoriginal Wassermann system.
The treatment of syphilitic serum with this salt can have a twofold benefit in the case of the original method of Wassermann;
namely, the removal of certain interfering serum components andthe removal or diminishing of the natural antisheep amboeeptor
present in the syphilitic serum.
In the method of Noguchi, there is no necessity for applying thebarium sulphate absorption. Noguchi recommends the use of unheated serum, hence the absolute amount of the serum employed isonly one-half of the absolute amount of complement. Eventually
an old serum may be anticomplementary and need inactivation, but
if the result is doubtful in this instance, a fresh serum from thesame patient may be secured and subjected to refixamination. In
this method there is no danger of introducing an amount of hemolytic amboceptor which is both unknown and uncontrollable, for the228

Barium Sulphate and the Serum Diagnosis of Syphilis.

reason that human serum is usually devoid of isolysin (antihuman
hemolytic amboceptor), and, if the latter is present, it never reaches
the strength which shows any effect upon the hemolytic system
employed.
We conclude, therefore, that the barium sulphate absorption is
to be recommended for the original method of Wassermann undercertain conditions, but that it is unnecessary for the antihuman
hemolytic system of Noguchi.