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Journal of Experimental Medicine

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A NEW AND SIMPLE METHOD FOR THE SERUMDIAGNOSIS OF SYPHILIS.'BY HIDEYO NOGUCHI, M.D.
(From the Laboratories of the Rockefeller Institute for Medical Research,
New York.)

Since the important discovery by Wassermann of serodiagnosis ofsyphilis, an enormous amount of work has been done, and to-day
its value as a diagnostic test is beyond dispute. The complexityof the original method and the requirement for a thorough knowledge of haemolysis and skill in serological technique have, however,been a great obstacle to its introduction into general practice. I
am convinced that, unless a great simplification of the method of
the Wassermann test can be devised, it will never attain the usefulness which it really deserves. Many workers have attempted tosimplify the method, but thus far, according to my view, without
success. 2'Received for publication, January I5, 909.
'Judging from my own observations which extended over oo cases, Bauer's
method (Deut. toed. Woch., I9o8, xxxiv, 698) is not only unreliable on accountof the inconstant content in natural anti-sheep amboceptor in human serum, but
it is also inconvenient, because it requires washed sheep's blood corpuscles andthe special apparatus of a laboratory. The method of Detre and v. Brezovsky(Wien. klin. Woch., 90o8, xxi, 1700-743) appears 'about equal in reliability
to the original, but in spite of its ingenious technique the use of washed horse
corpuscles interferes with its usefulness outside of a laboratory.
A so-called simple method of serum diagnosis of syphilis recently publishedby Tschernogubow (Berl. klin. Woch., 908, xlv, 2I7) is of a different ordersince he states that the complement contained in the blood of patients is sufficient in quantity to reactivate anti-human amboceptor and cause haemolysis of
the same blood. No mention is made of the source and strength of the antihuman amboceptor referred to. Tschernogubow's method relies upon an unknown quantity of complement which varies in different specimens of humanblood. As the complement and erythrocytes deteriorate rapidly, his method is
inapplicable to old specimens. There is also no direct way of testing the inhibitory action of the antigen alone. Anti-human amboceptor derived from
immunized rabbits is reactivate only by large amounts of human serum, which
renders the method inconvenient. Finally the idea of Hecht (Wien. klin. Woch.,392

Hideyo Noguchi.

393

All the methods so far proposed are indeed suited only for the
fully equipped laboratory possessing centrifuge, thermostat, waterbath, etc.
I have, I believe, been successful in overcoming the difficulties ofthe situation by perfecting a new and simple method of syphilis
serodiagnosis. This method offers definite advantage in point ofdelicacy and simplicity for the experimental serologist; and thereagents to be employed can be prepared for and put within reachof the practitioner so that the test may be made by any physician
who is in the habit of making clinical laboratory tests, such as the
Widal test, hemoglobin estimation, etc.
PRINCIPLES OF THE METHOD.

The new method is based upon the same principle as Wassermann's and utilizes the Bordet-Gengou phenomenon of complementfixation to determine the presence of the syphilis antibody in a given
specimen of blood serum or cerebrospinal fluid. It differs, however, from the Wassermann method in using an anti-human hemolytic system instead of an anti-sheep hemolytic system. This difference may at first seem of trifling importance but it really is verysignificant since it affects the accuracy, ease and reliability withwhich the test can be applied. Wassermann's original method, it
may be added for sake of clarity, is subject to an error arisingfrom the presence in human serum of a varying amount of natural
amboceptor capable of being reactivated by guinea-pig's complement. Many specimens of human serum contain in o.I c.c,, whichis the quantity usually employed in a test for complement fixation,as many as twenty units of anti-sheep amboceptor, while some specimens are entirely devoid of this amboceptor. I found by actual
experiment that four units, but not two units, of the natural antisheep amboceptor prevent entirely the detection of one unit ofsyphilis antibody. When eight units of natural anti-sheep amboceptor and two units of syphilis antibody are brought together,
19o8, xxi, 1742) that the native hemolytic property of human serum alone may
be used as an hemolytic system in combination with washed sheep's corpuscles
is impracticable, as the amount of human complement and amboceptor is sovariable as to make the test unreliable without a special standardization of such
serum.

394

Serum Diagnosis of Syphilis.

the test remains completely negative. In other words, by themethod of Wassermann the presence of syphilis antibody may be
missed although in the absence of the natural anti-sheep amboceptor
it would be readily detectable. This defect in the complement fixation system is not only inherent to the Wassermann method but
also to all those which employ foreign blood corpuscles for whichhuman serum contains natural hemolytic amboceptors capable ofbeing reactivated by the complement employed in the test.
I believe that the method which I have worked out eliminatescompletely this source of error, because human blood corpuscles
are used as the hemolytic indicator in combination with humanserum, so that as no foreign natural haemolytic amboceptor is
accidentally present, its effects are excluded. The present methodof the complement fixation test is carried out under conditions ofdefinite and uniform sensitization of the blood corpuscles, thus
enabling one to detect the presence of even a fractional part of one
unit of syphilis antibody in a given specimen. It is impossible tomiss one or more units of the antibody, which happens not infrequently with the Wassermann and similar methods.
DESCRIPTION OF THE METHOD.

In order to carry out the present method of diagnosis it is needless to say that the serum of the patient under question must be
obtained. The patient's serum is then subjected to the usual complement fixation test with the reagent to be described immediately
below and according to the technique specially adapted for thepresent method. I have developed so far two different ways ofapplying the test. In one the reagents are employed in liquid form
and in the other, the reagents are used in a dried state on filter
paper. The first way of making the test is adapted to general biological laboratories, and the second is adapted especially for theclinical laboratory. The descriptions which follow immediately
refer to the laboratory method, but they do not differ essentiallyfrom those for the clinical laboratory except that the reagents
employed are in the two different forms mentioned.

Hideyo Noguchi.

396

REAGENTS REQUIRED FOR THE METHOD.

The following reagents are required for making a test by thepresent method:
I. Anti-human hcemolytic amboceptor prepared in rabbits by injecting them five or six times into the peritoneal cavity with increasing doses (up to 20 c.c.) of washed human blood corpuscles,
allowing five days interval between each injection. The serum iscollected from the immunized animal eight or nine days after thelast injection. The titre must be stronger than o.oI c.c. for complete haemolysis.3 I now use a serum of o.ooI c.c. titre.
2. Complement. Fresh guinea-pig serum.
3. Antigen. Alcoholic extract of organs or crude preparationsof lecithin. To prepare the alcoholic extract of organs one partof mashed tissue (liver, kidney) is extracted with ten parts ofabsolute alcohol for several days at 370 C., filtered through paper
and the filtrate concentrated to about one-third of its volume, andthe fluid preserved. To prepare the lecithin solution for antigen,
0.3 gram is dissolved in 50 c.c. of absolute alcohol and then shaken
with 50 c.c. of physiological salt solution and filtered. The filtrate
must be clear.
4. Suspension of human blood corpuscles. This is prepared bymixing one drop of the blood of a normal person 4 with 4 c.c. ofphysiological salt solution.
5. The serum to be tested. About ten drops of blood from apatient are collected in a small test tube. The clear serum separated from the clot is employed for the test.
METHOD OF MAKING THE TEST.

I. Take six clean test tubes (size Io cm. x I cm.). In the firsttwo of these, place one drop from a capillary pipette (or tube ifnecessary) of the patient's serum which is to be tested. In eachof the second two tubes (which are to serve as controls) put one
drop of the serum of a syphilitic case known'to give the positive'Complete haemolysis of c.c. of human blood suspension ( drop) in thepresence of 0.025 c.c. of guinea-pig's fresh serum.
'The suspension can also be prepared with the patient's blood, but it must
be used only in combination with the serum of the same patient.

Serum Diagnosis of Syphilis.

396

reaction. In each of the third pair of tubes (also controls) put
one drop of the serum of a normal person. Now to each of thesix tubes add I c.c. of the suspension of human blood corpuscles
and 0.04 c.c. of fresh guinea-pig serum as complement. Lastly
into one of each of the three foregoing pairs of tubes put one
drop of the antigen solution from a capillary pipette. The second
tube of each pair receives no antigen.
2. After being well mixed by shaking, the six tubes are incubated at 370 C. for one hour.
3. At the end of the incubation, add two units of anti-humanamboceptor to each tube and mix well by shaking. Incubate thetubes for two hours longer at 370 C. Read the reaction from time
to time for the next ten to twelve hours during which the tubes are
kept at room temperature.
The complete test is shown in the following chart.
CHART I.

Test for Diagnosis.

Control tube without antigen foreach test.

a. Patient's serum.
b. Human blood
suspension.
c. Complement.

Determinati ve
tube containing
antigen.

a.

6

I' st Control Set.*
Test with a Known Syphilitic Serum (Positive
Reaction).

2d Control Set.t
Test with a Normal
Serum (Negative Reaction).

a'. Positive luetic
serum.

O b. Human blood
suspension.
c. Complement.
ditto.

a". 1Cditto.

d. Antigen.

d. Antigen.

a'.

ditto.

0b.

C.
.
d. Antigen.
Incubation at 370 C. forat". Normal serum.
6. Human blood
suspension.
e. Complement.

hour.

Anti-human hemolytic amboceptor to all tubes.
Incubation at 37 °0 C. for two hours longer, then at room temp.

*When many specimens of syphilitic sera are subjected to simultaneous
tests this set of controls is scarcely necessary, as we usually find a good positive serum among them.
t This control set may be made without the addition of any human serum.

It is necessary to begin the test with one drop of the patient's
serum. If the reaction is negative the test should be repeated withtwo drops. Sera are not infrequently met with, however, in which
two drops are inhibitory without the presence of antigen. On thisHideyo Noguchi.

397

account it is undesirable to use two drops as routine. Usually
one drop reveals the presence of the specific antibody if it ispresent at all, and further test becomes almost unnecessary.
READING OF THE REACTION.

A negative reaction is indicated by complete hemolysis in bothtubes irrespective of the presence of the antigen. On the other
hand, a positive reaction is shown by complete absence or partial
inhibition of haemolysis in the tube containing the antigen and complete hemolysis in the tube without the antigen.
Complete hemolysis in the negative control takes place usually
within one hour or -sometimes earlier. When the reaction isstrongly positive, as it should be with the positive control set, thetube with the antigen remains absolutely free from any trace of
haemolysis even after a few days' standing at room temperature.
In such a case the erythrocytes are strongly agglutinated and remain adherent to the bottom of the test-tube, leaving above a perfectly clear medium. In moderately positive cases there is a traceof hemolysis, while the varying degree of positive reaction canreadily be measured by the amount of the hemoglobin liberated in
the medium and especially by the amount of intact red corpuscles
remaining in the fibrin shreds or whitish stroma masses. It shouldbe mentioned that the tubes containing the antigen undergo hmolysis somewhat more slowly than those without antigen, but complete
haemolysis must occur in both tubes of the negative control set,
before the reaction of the other sets of tests is read. It is also
necessary to note that every tube without antigen, irrespective of thenature of the specimen of blood serum therein contained, must
undergo complete, or at least almost, complete hemolysis before
the final reading of reaction is made. In cases in which tubes without antigen do not undergo hemolysis the test must be repeatedwith a smaller quantity of the human serum in order to obtain adefinite result.
SIMPLIFIEDMETHOD FOR THE CLINICAL LABORATORY.

The method so far described is much simpler than the original
test of Wassermann and can be quickly and easily carried out in any

398

Serum Diagnosis of Syphilis.

laboratory where experimental work with serum is constantly being
conducted. I have, moreover, been able so to modify the methodsof handling the various factors in the test that it should be possible to carry it out' with accuracy and dispatch in any clinicallaboratory.
These further modifications consist essentially indrying the reagents on paper and standardizing them. In this form
they can be prepared on a large scale by commercial biologicallaboratories under supervision of a competent serologist, and placedon the market within ready reach of physicians. My experiments
have shown that it is possible to do this with great accuracy andthat the preparations so made are stable when kept under ordinary
conditions. In the following paragraphs I will describe the methodof preparing the filter paper reagents and the mode of employing
them for the test.
Anti-human amboceptor slips. The serum of immune rabbits is taken up with filter paper and quickly dried (a few
hours) by a current of air at low temperature (below 20o C.). If
the immune serum is not very strong-the titre o.oI c.c. for example-the serum should first be concentrated to one-third of its
volume by means of a current of air before the paper is impregnated. This is necessary in order to secure a slip of small dimension for convenient use.) After complete desiccation, the impregnated paper is cut into pieces of equal dimensions, each one beingof such a size as to contain two units of the amboceptor.
Complement slips. A rather thick filter paper is impregnated with fresh guinea-pig serum, similarly dried and cut into
pieces of equal size. The activity of the complement slip must be
titrated and compared with the fresh complement. The activityof the slip-complement may vary somewhat with every preparation,but one slip should possess such an activity as to correspond withthat of 0.04 c.c. of the fresh guinea-pig serum.
Antigen slips. A crude preparation of lecithin of previously
known antigenic value is dissolved in ether and similarly taken up
into filter paper. It is also easy to impregnate the paper with thealcoholic organ extracts (such as from the liver of a congenitallysyphilitic fcetus), but a preliminary concentration of the extract bymeans of a current of air is necessary in this case. After impreg-

Hideyo Noguchi.

g399

nation and complete desiccation, the impregnated paper is cut into
equal parts, each being of such a size as to contain enough antigen
for one tube.
These three reagents, the anti-human hamolytic amboceptor,
guinea-pig complement and antigen, in the impregnated filter papers,
can be kept indefinitely at room temperature in a dry place. I haveadopted as the dimension of each slip the size of about 5 mm. by
5 mm. I t is understood that preliminary tests should always be
made with the slips to prove activity and strength.
I n employing the filter paper slips, they are dropped by means offorceps into the test tubes already containing the human blood suspension and patient's serum, in the order and at the intervals already stated for the respective reagents (complement, antigen, amboceptor) in the description for the method of making the test
(Chart I). It is necessary to shake the tubes a few times at intervals in order to insure proper solution of the reagents. The incubation may be carried out perfectly well in the vest pocket if athermostat is not at hand. Confusion is to be avoided by carefullylabelling the various tubes. Each of the pairs of tubes making
up, respectively, the test, and the two control sets, may conveniently
be held together by a rubber band. There can be no confusion
within one pair of tests since one of the two tubes contains one
more slip (antigen) than the other.
I should advise any one contemplating the use of the method tomake a few preliminary trials comparing cases definitely syphilitic
with normal individuals before undertaking the diagnosis of casesof unknown nature. This would also have the advantage of accustoming the physician to the order of the procedures and thereactions and serves as a test of the reliability of the reagents.
RESULTS.

I n all, four hundred and sixty-five specimens of serum havebeen examined by the method above outlined. One hundred andfifteen of these were examined at the same time by the original
method of Wassermann. The results are tabulated as follows:400

Serum Diagnosis of Syphilis.
TABLE I.5

Amount of Treatment.
Method.*0
W

Reaction.
Primary Lues.
Secondary Lues.
Manifest.
Latent.
Tertiary Lues.

±

Manifest.
Latent.
Suspects.
'on-syphilitic.

_

_

r

An

Slight.

~
ArW
W

N

o +

6o

4

2

18

2 20 00
0 1 00

4

I

3

0

3

4

0

IN

0
I

4
4

Moderate.

Fairly Well.

W

W

N

0

W

N

+

o
0
0

Weil.

I

2

4

2

3 0
I

I

I

I

8 o 8 0
6 I
52

I

I

I

12 21 7x60
O 5 0o5* [V = Wassermann's test; N = the present test.

+

+

6 0

N

2

0

2

2

t

2

I 5

4

7

I
2

Summarized the results are as follows:
Of 7 cases of primary syphilis the Wassermann test was positivein 5, the present test in all.
Of 27 cases of manifest secondary syphilis the Wassermann test
was positive in 23, the present method in all 27.
Of I2 cases of latent secondary syphilis the Wassermann test was
positive in 6, the present method in 9.
Of I8 cases of manifest tertiary syphilis the two methods agreed
and were positive in 17.
Of I8 cases of latent tertiary syphilis the Wassermann test was
positive in iI, the present method in I4.
Of ii untabulated cases of tabes dorsalis only 3 were positive
with the Wassermann test and all were positive with the present
test.
Of 28 cases suspected of syphilis or of syphilitic origin, the Wassermann test was positive in 6, the present method in 21.
Concerning certain points in the manner of employing the various
reagents in testing the cases above reported, I may state that about
one-third of all the cases were examined with liquid preparations
The cases recorded in this table were derived chiefly from the clinics
of Dr. Victor C. Pedersen of the New York Hospital and House of Relief,
Dr. Howard Fox of the Vanderbilt Clinic, and Dr. J. A. Fordyce (through Dr.
Homer F. Swift) of Bellevue Hospital. Ten of the suspected cases belong to
Dr. Jacob Heckmann of the German Hospital Dispensary, and will be reported
by him from a clinical standpoint in a later publication. It is my pleasant duty
to acknowledge my indebtedness to these gentlemen for their assistance infurnishing me with the clinical material.

Hideyo Noguchi.

401

of complement, antigen and amboceptor, while a second third were
tested in a similar manner except that the complement and amlboceptor were first dried and pulverized before they were dissolved insaline solution for use. The remaining third of the cases were examined by the reagent paper method. The results obtained by thislast method were perfectly reliable and satisfactory. It is needlessto say that every one of these tests had been carefully controlled by
the use of liquid reagents and by the regular Wassermann test.
In comparing the results obtained by the original Wassermann
method with those obtained from the simple method here described,
it should be pointed out that cases which were negative to the
Wassermann and weakly or often quite strongly positive to thepresent method were met with. The causes of the observed differences were found in the existence of a large amount of natural
amboceptor for sheep's blood corpuscles in such specimens of humanserum. No disagreement in the result between the two methods
was found with strongly positive specimens (containing more thaneight units of syphilis antibody in o.I c.c.) or with weak specimens
devoid of the natural amboceptor.
I would finally draw attention to the value of the test as a guideto the treatment of cases of syphilis. It appears that with the progress of the cure the symptoms of the disease not only abate, but the
blood reaction grows weaker until ultimately when the cure has
been established the reaction can no longer be obtained. If experience supports this result, the duration of the antisyphilitic
treatment and its efficacy may become subject to quantitative control,
The advantages which would accrue from such a measure are
obvious. I mean to pay strict attention to this important therapeutic
point and shall hope to report on a much larger series of cases in thenear future.