野口英世著

Journal of Experimental Medicine

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SUBLIMATE AND THE SERUM DIAGNOSISOF S Y P H I L I S . *BY H I D E Y O N O G U C H I AnD J. B R O N F E N B R E N N E R .

(From the Laboratories of the Rockefeller Institute for Medical Research,New York.)

According to the studies of those who have been employing theantisheep hemolytic system of Wassermann, the serum reaction ofsyphilis is apt to disappear suddenly or become very weak shortly
after the commencement of mercurial treatment, while the clinicalaspect of the patient shows no improvement. This rapid disappearance of the reaction, although not very frequently encountered,renders the value of the Wassermann reaction quite uncertain as aprognostic measure in the treatment of syphilis.
The cause of this phenomenon has not been fully explained. As
it has been observed only when the antisheep system is being used,
one is justified in seeking the source of this irregularity in the fluctuation of the amount of natural antisheep amboceptor in the serumof the patient during the treatment. A serologist should expect acertain degree of irregularity due to the use of an heterohemolytic
system through variable and inconstant' fluctuations of the natural
hemolytic amboceptors in the patient's serum. By adding immune
hemolytic amboceptors, he can produce experimentally conditions
which will cause a positive serum to give a negative reaction. This
result is always obtained when the reaction is not very strong.
In practice it has been repeatedly demonstrated that the antihuman hemolytic system of Noguchi reveals the syphilitic antibody
where the Wassermann system fails on account of the presence of
natural amboceptor. Any one who has used both methods adequately must have concluded that the Noguchi system gives a moredelicate and constant result with the serum of the patient undertreatment. Pedersen, who utilized the antihuman system of Nogu* Received for publication, September 21, 1910.

210

Hideyo Noguchi and J. Bronfenbrenner.

211

chi, in following up the effects of treatment found a perfect harmonybetween the intensity of the reaction and the clinical course of thedisease during the treatment.
Efforts have been made by some investigators to discover whether
or not the sudden disappearance or weakening of the reaction is the
direct effect of the mercury upon the syphilitic antibody. Theirresults are contradictory, as some claim to have obtained a negative
reaction with a positive syphilitic serum by mixing it with soluble
mercurial preparations in test tubes, while others observed no sucheffects.
We have made some experiments to ascertain what effects asoluble mercurial preparation will have on the syphilitic antibody invitro. _As mercurial preparation, we have chosen mercuric bichloride; and for the fixation test, the system of Noguchi.
The effect of bichloride of mercury was determined upon ( I )red blood.corpuscles, (2) complement, (3) antihuman amboceptor(rabbit), (4) syphilitic antigen (aceton-insoluble tissue lipoids ofhuman liver), and (5) syphilitic antibody. The results obtained
are given briefly in what follows.
Hemolytic Power of Bichloride of Mercury.--In a series oftubes, each containing one cubic centimeter of a I per cent. suspension of washed human corpuscles, varying amounts of bichloride ofmercury in suitable concentrations were added and the mixtures
were incubated for two hours at' 37 ° C. The results are as follows :Bichlorideof m e r cur,] in g r a m s .
O.OOIo.ooo3o.oooz
0.oooo3
o.ooooz
0.oo00o3
0.ooooo25
0.00ooo2
0.0ooo015
o.oooooi
o.oooo0o7

No hemolysis (precipitate, discoloration)
3o per cent. hemolysis (precipitate)ioo per cent. hemolysis (precipitate)ioo per cent. hemolysis
zoo per cent. hemolysis
ioo per cent. hemolysis
ioo per cent. hemolysis
95 per cent. hemolysis
9o per cent. hemolysis
No hemolysis
No hemolysis

The hemolytic power of bichloride of mercury was considerably
reduced by mixing it with o.o 5 cubic centimeter of guinea pig complement or with two units of the amboceptor serum on paper.

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Sublimate and the Serum Diagnosis of Syphilis.

Bichloride of mercury in grams,
O.0OO005
0.000003
0.0000025
0.000002
0.0000015
0.000001

0.05 c.c. complement (incubatedi hour before addition ofcorpuscles),IOO per cent. hemolysis
N o hemolysis
N o hemolysis
N o hemolysis
N o hemolysis
N o hemolys,is2 units amboceptor (paper) (incubated x hour before addition of corpuscles).
Ioo per cent. hemolysis
IOO per cent. hemolysis
100 per cent. hemolysis
60 per cent. hemolysis
N o hemolysis
N o hemolysis

Thus, the original strength, 0.0000025 , for complete hemolysis
became 0.000005, and the non-hemolytic quantity, o.oooooI, became 0.000003 after the bichloride had been digested with 0.05
cubic centimeter of complement. It is evident, therefore, that 0.05
cubic centimeter of complement absorbed the differences during one
hour's contact with the sublimate. The absolute quamity ofbichloride of mercury absorbed by this amount of serum is about
0.000002 gram. The amboceptor serum interfered in a lesser
degree.
The hemolytic power of bichloride of mercury was not noticeably
diminished by the antigen, which was used in doses of 0.0003 gram.

Effects of Bichloride of Mercury upon Complement and Amboceptor.--In order to detect any possible anticomplementary propertyof bichloride of mercury, 0.005 cubic centimeter of guinea pig complement was mixed with varying quantities of bichloride of mercury
and the mixtures were incubated a t 37 ° C. for one hour. At theend of incubation the corpuscle suspension and two units of amboceptor were introduced, and the mixtures were again incubated at37 ° C.
]Bichloride of merc m y in grams.
0.0000025
o.0000oi5
o.oooooT
0.00o0006
0.o000003
control

Complement o.oo5-{Amboceptor (two units).
Complete hemolysis
Complete hemolysisComplete hemolysis
Complete hemolysis
70 per cent. hemolysis
70 per cent. hemolysis

Complement 0.oo5-kNo amboceptor (control).
Complete hemolysis
N o hemolysis
N o hemolysis
N o hemolysis
N o hemolysis
N o hemolysis

The experiment indicates that bichloride of mercury has no anticomplementary effect in the quantities used. On the contrary, thehemolysis was more complete and proceeded more rapidly in its

Hideyo Noguchi and J. Bronfenbrenner.

213

presence. This is due to the injurious effect of bichloride of mercury upon the corpuscles, thus reducing the corpuscular resistanceto the hemolytic action brought about by the complement and amboceptor.

Effects of Bichloride of Mercury upon the Syphilitic Antigen andAntibody.--To determine the effect of bichloride of mercury upon
the antigen, it was mixed with the latter and incubated for one hour
at 37 ° C. before introducing the syphilitic serum and complement.
After the incubation, the syphilitic serum and complement wereadded and incubated, as usual. Amboceptor and corpuscles wereadded and the whole was again incubated.
To determine the effect of bichloride of mercury upon the syphilitic antibody, it was mixed with the latter and incubated for one
hour at 37 ° C., then, at the end of incubation, the complement andantigen were introduced, the remaining procedures being the sameas usual.
The amount of complement employed was 0.05 cubic centimeter;that of amboceptor, two units; that of antigen, 0.0003 gram ofaceton-insoluble lipoids; that of syphilitic serum, 0.02 cubic centimeter; and that of corpuscles, o.I cubic centimeter of a IO percent. suspension. The total volume of fluid in each tube equalled
one cubic centimeter.
The foregoing experiments show that bichloride of mercury inthe highest non-hemolytic quantity (0.0000025 gram) failed to remove, to a noticeable degree, the syphilitic antibody, and the reaction in two strongly positive sera remained unchanged. In twoweaker sera there was a little more hemolysis than in the control
tubes which contained no bichloride of mercury. This slighthemolysis may be explained, ( I ) by the removal of some of theantibodies by the bichloride, (2) by the injury inflicted by the bichloride upon the red blood corpuscles, rendering these more readily
hemolyzable by the complement and amboceptor introduced afterward, or (3) by the combined action of ( I ) and (2). O f these
possibilities, the second is the more probable, this assumption being
supported by the fact that, in the controls in which no bichlorideof mercury was present, and in which no hemolysis occurred at
first, there was a trace of hemolysis after these had stood for six

214

Sublimate and the Serum Diagnosis of Syphilis.
•-.

"sanoq~..

~. ~ 9U.~Huu

O~~ZZ,~._=0 0 0•sznoq o g+.~6~ oZoZoZoZ~

Nod0o~mZz000000

Z Z Z Z Z Z

8 ¸[-~~•0000000

ZZZZZZZ. "szno~•>,~~,.-1<o~oo[•~=ozzz~= o • • •O~o ~ Z0Z Z0 0"slnoq ogo~ o oo oZZ~ZZ.

6do.uo N-tqtqm ~nI~qV~

ooo"uo!:~-!q!Itu! o ~ n l o s q v

8~h0oo o o~0 °°°°~
N~00000~0000
0000

dd6doIIHideyo Noguchi and J. Bronfenbrenner.

215

hours or longer, showing that the complement was not completelyfixed. The same is true also in the experiments relating to theeffect of sublimate upon the antigen. When working without
proper cohtrols (such as determine the effects of non-hemolytic
doses of bichloride of mercury upon the complement-amboceptor
hemolysis), one is apt to consider the above experiments as constituting evidence of the removal of the syphilitic antibody by thesublimate. It may be added here that the strongly positive sera
(No. I and No. 2) contained only about two units of syphilitic
antibody in 0.02 cubic centimeter. In test tube experiments it seems
to us that the only way to determine the slightest possible effect of
sublimate upon syphilitic antibody is to test the action of the highest non-hemolytic quantity of bichloride of mercury upon the smallest detectable number of antibody units. This was done in ourpresent determination.
Whether the same results are obtainable with the ,antisheep
hemolytic system is another problem. If the resistance of sheep
corpuscles to the hemolytic effect of bichloride of mercury is greater,thus permitting the use of a larger amount of the mercurial salt,a more distinct effect might be secured. But the work of Sattaand Donati shows it to be about the same. One might imagine thatthe hemolytic effect of the mercurial salt can be prevented byadding certain indifferent proteins to the mixture, but, unfortunately, according to our recent studies, the indifferent proteins canthemselves saturate the fixing affinity of the mixture of syphilitic
serum and antigen and render the positive reaction negative.
The test tube experiments do not, however, exclude the possibility that bichloride of mercury acts in the body upon the syphilitic
antibody. In the body the sublimate circulates constantly and may
remove the syphilitic antibody little by little. Besides, the richprotein content of the body fluids, especially the blood serum,protects the red blood corpuscles from the injurious effect of bichloride of mercury to a far greater degree than the conditions in vitro
permit. The question is not adequately answered by means oftest tube experiments. It is well to recall here that the administration of the powerful spirochaeticide, dioxy-diamido-arsenobenzol,

216

Sublimate and the Serum Diagnosis of Syphilis.

of Ehrlich and H a t a can lead to the disappearance of the syphilis
reaction in a comparatively short period, yet this preparation contains no mercury. The disappearance of the reaction under themercurial treatment may, therefore, be due, not to an influence on
the antibody, but to a destruction of the spirochaete.