野口英世著

Journal of Experimental Medicine

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A H O M O H E M O L Y T I C SYSTEM F O R T H E S E R U M
DIAGNOSIS OF SYPHILIS.
BY HIDEYO NOGUCHI, M.D.
(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, March 20, 1918.)
There is little doubt that the elimination of the use of guinea pig
complement from the serodiagnostic procedure is a great advancetowards the simplification of this important reaction, for that element has to be used while it is perfectly fresh, necessitating themaintenance of guinea pigs in the laboratory. Attempts to preserve
the activity of the complement, either by means of desiccation ~ orhypertonization, 2 have been only partially successful, and it deteriorates in a comparatively short time. The best preservative for guinea
pig complement is that recommended by Rhamy, 3 who found that
sodium acetate in appropriate concentration keeps the complement
active for several weeks.
Some years ago HechO proposed the utilization of the natural antisheep hemolysin and complement in the fresh serum of patients, but
the difficulty in this method lies in the fact that in some sera there isno natural hemolysin or too little to produce hemolysis, even in
the control tubes without the antigen. On the other hand, somesera contain a considerable excess of the natural anti-sheep hemolysin.
Granting that the insufficiency of the natural anti-sheep hemolysin
can be remedied by adding an adequate quantity of a non-syphilitic
1Noguchi, H., On the influence of the reaction and of desiccation upon oposo
nins, J. Exp. Med., 1907, ix, 455; Serum diagnosis of syphilis, and the butyricacid test for syphilis, Philadelphia and London, 1st edition, 1910.
2 Austin, F. D., A new method for preserving Complement for making the
Wassermann or Noguchi blood-test, J. Am. Med. Assn., 1914, lxii, 868.
3 Rhamy, B. W., Preservation of complement. A preliminary report, J. Am.
Med. Assn., 1917, lxix, 973.
4 Hecht, H., Eine Vereinfachung der Komplementbindungsreaktion bei Syphilis,
Wien. klin. Woch., 1909, xxii, 338.
43

44

SERUM DIAGNOSIS OF SYPHILIS

serum containing enough hemolysin, the test still calls for the washedsheep corpuscles. In other words, having eliminated the use ofguinea pig serum as complement, we still have to depend upon the
sheep corpuscles for an indicator of hemolysis.
In ordinary times there should be no difficulty in obtaining theguinea pig complement or sheep blood corpuscles, and it would
be immaterial whether one used the anti-sheep hemolytic systemor the anti-human hemolytic method as advocated by the writer ~and accepted by many serologists, including those in the UnitedStates Army and Navy. Perhaps it may not be amiss to call attention here to the fact that the anti-human hemolytic system of
the writer is not limited in its use to unheated sera but applies
equally to inactivated sera, the only differences between the examination of fresh and inactivated sera being that only 0.02 cc. of the former
is required, instead of 0.08 to 0.1 cc. of inactivated specimens, andthat in the case of inactivated sera not only the acetone-insoluble
fraction of tissue lipoids, but also any properly titrated alcoholic
extracts may be used as the antigen. 6 With fresh sera emphasis has
been placed on the fact that the crude alcoholic extracts or those alcoholic extracts to which cholesterol has been added are to be avoided,
as in this combination there is apt to occur a non-specific proteotropic
complement fixation. 7 It is therefore essential that only the acetone-insoluble fraction of tissue lipoids should be used in combination with fresh sera. With these points in view there should be no
confusion as to the extent of applicability of the test to fresh andinactivated sera. 6
There is another possibility, namely that by eliminating the useof the guinea pig complement from the anti-human hemolytic system
the technique and the material for the serodiagnosis of syphilis canbe greatly simplified. That this is the case is shown below. Efforts
to introduce similar methods have already been made by Tscher|s Noguchi, A new and simple method for the serum diagnosis of syphilis, J.
Exp. Med., 1909, xi, 392.
6 Noguchi, Serum diagnosis of syphilis, and luetin reaction, together with the
butyric acid test for syphilis, Philadelphia and London, 3rd edition, 1912.
7 Noguchi, On non-specificcomplementfixation, Proc. Soc. Exp. Biol. and 2~led.,
1909-10, vii, 55.

ninE:CO NOGUCm

45

nogubow, s E m e r y / Butler and Landon, x° Myer, n and Thompson, ~all employing h u m a n complement. A brief review of these methods
will be given later.

Principle of the Method.

I t is well known t h a t complement is present in every fresh serum
and t h a t the q u a n t i t y m a y sometimes vary. In the serodiagnosis ofsyphilis, whether by the anti-sheep system of Wassermann or the antih u m a n system of the writer, the serum of the guinea pig is chosenbecause of its richness in complement and also because guinea pig
complement is probably more readily fixed 13by the antigen-antibody
combination than the sera of other animals, such as the horse, rabbit,
sheep, pig, etc. ~4,~5 As to the comi~lement in fresh human sera, there
are not m a n y d a t a regarding its action upon h u m a n corpuscles. I t
is understood t h a t h u m a n sera exert no hemolytic action upon h u m a n
corpuscles, especially when there is no isohemolysin. B u t upon the
addition of a sufficient quantity of the anti-human hemolytic amboceptor the complement dissolves the human corpuscles just as guinea
pig complement does when added to the suspension of h u m a n corpuscles and the specific anti-human hemolytic amboceptor.
s Tschernogubow, N. A., Ein vereinfachtes Verfahren der Serumdiagnose bei
Syphilis, Deutsch. reed. Woch., 1909, xxxv, 668.
9 Emery, W. d'E., Clinical bacteriology and ha~matology for practitioners,
Philadelphia, 4th edition, 1912.
10Butler, C. S., and Landon, W. F., A technic for the absorption test forsyphilis using human complement, U. S. Nay. Med. Bull., 1916, x, 1.
11 Myer. S. B., A complement-fixation test for syphilis using human complement, U. S. Nay. Med. Bull., 1917, xi, 175.
12Thompson, L., Complement fixation in syphilis, with a preliminary reportof a new technic, Am. J. Syph., 1917, i, 555.
13 Noguchi, H., and Bronfenbrenner, J., Variations in the complementary activity and fixability of guinea pig serum, J. Exp. Med., 1911, xiii, 69.
t4 Noguchi, Non-fixation of complement, Proc. Soc. Exp. Biol. and Med., 190910, vii, 14.
15 Noguchi and Bronfenbrenner, The comparative merits of various complements and amboceptors in the serum diagnosis of syphilis, J. Exp. Med., 1911,
xiii. 78

46

SERUM DIAGNOSIS OF SYPHILISThe only difference between the action of the human and the guinea
pig complement lies in the fact that the former requires more antihuman hemolytic amboceptor to render it active against the human
corpuscles than the latter. The relative lytic values of the human and the guinea pig complement are shown in the following
experiments.

Comparative Complement Values of the Human and the Guinea Pig Sera
R'elative to the Anti-Human Hemolytic Amboceptorand Human Corpuscles.
0.04 cc. of guinea pig complement was put in each of a number oftubes, together with 1 cc. of a 1 per cent suspension of human corpuscles. Varying quantities of the.anti-human hemolytic amboceptor(immunized rabbit) were added to the tubes and the results read
after 30 minutes at 37°C. in a water bath thermostat. The results
are shown in Table I. The experiment shows the titer of the antiTABLE I.

Titration of the Anti-Human Amboceptor with Guinea Pig Complement.
Amount of serum toeach tube.*

Anti-human hemolytic immune serum.
No. 633 (rabbit).

No. 634 (rabbit).

Complete hemolysis.

Complete hemolysis.

CO.

0.01
0.007
0.005
0.004
0.003
0. 0020.00150.001
0. 00070.0005
O. 0004
0.00030.0002
0

gt

gt

~c

cc

tg

t~

gc

~c

c~

tc

cc

~c

*~

tc

~c

~C

*c

*g
cC

~¢

Partial hemolysis.
No
ct

~C

g~

~c

gg

gc

Partial hemolysis.
No
"
c~

cc

* E a c h t u b e c o n t a i n e d g u i n e a p i g c o m p l e m e n t 0.04 cc. a n d 1 p e r c e n t h u m a n
c o r p u s c l e s u s p e n s i o n 1 cc.

47

HIDEYO NOGUCHI

h u m a n hemolytic immune serum No. 633 to have been 0.0007 cc. andt h a t of No. 634, 0.0005 cc. in the presence of 0.04 cc. of guinea pig
complement. 0.1 cc. of the guinea pig serum alone was itself somewhat hemolytic.
F r o m the results recorded in the first p a r t of Table I I it appears
t h a t in the presence of 0.1 cc. of a fresh h u m a n serum at least 0.005
TABLE II.
Titration of the Anti-Human A mboceptor with Human Complement.
Serum 3 +amboceptor.*

Result.

CC.

CC.

0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

+ 0.1
+0.05
+0.03
+O.O2
+ 0.01+ 0.005+0.0O3+0.002+ 0.001

Complete hemolysis.

0.5 + 0.02
0.3 + 0.02
0.2 + 0.02
0.1 + 0.02
0.05 + 0.02
0.02 + 0.02

Complete hemolysis.

c¢

t~

~c

cc

~c

c~

cc

g~

Considerable "
No
~g

cc

~c

~c

Slight hemolysis.
No"* Human serum as complement. 1 per cent human corpuscle suspension 1 cc.
in each tube.
cc. of the anti-human a m b o c e p t o r No. 633 was needed to produce
complete hemolysis; t h a t is, a b o u t seven times the a m o u n t requiredin the case of guinea pig complement. I n other words, the activityof h u m a n complement is nearly one-seventh t h a t of the guinea pig
serum.
In the second p a r t of the table it is shown t h a t 0.05 to 0.02 cc. ofthe same f r e s h h u m a n serum was unable to cause complete hemolysis
even in the presence of 0.02 cc. of the amboceptor N o . 633, which isan equivalent of t h i r t y minimal lyric doses when calculated on thebasis of 0.04 cc. of the guinea pig complement. I t is evident t h a t

48

SERUM DIAGNOSIS OF SYPHILIS

the amount of amboceptor required to dissolve the human corpusclesin the presence of human complement is many times that necessary
with guinea pig complement.
The next point is to determine whether or not average fresh humanserum contains enough complement to produce complete hemolysis.
Upon this fact depends the possibility of utilizing the human cornTABLE III.

Quantitative R e l a t i o n between Human Complement and A n t i - H u m a n Amboceptor.
Human Serum 0 . 0 5 c c .

Human serum 0 . 1 c c .

~6
Amboceptor

cc.

Amboceptor 0 . 0 1

cc.

Amboceptor
0.005 co.

Amboceptor

0.01 cc.

Considerable hemolysis, Complete hemolysis No hemolysis. Complete hemolysis.
*c4¢
No hemolysis.
No hemolysis.
No hemolysis.
g4
gg
Considerable hemolysis. Complete hemolysis
Complete hemolysis.

1

2
3
4
5
6

7
8
9
10
11
12
13
14
15
16
17
18
19
20

0.005

~4

44

44

lg

~4

gc

~C

~4

~4

44
~4
Complete hemolysis.
~44~Considerable hemolysis.
No hemolysis.
No hemolysis.

c4

Lg

C4

~¢

4g

4L

LC

4~

g4

~4

gg

Considerable hemolysis. Complete hemolysis,
Almost complete "Considerable

"

~4

~4

Complete hemolysis.

~4

~4

¢4

L~

~4

~4

*c

~4

~4

g¢

g4

~4

4~

~4

~4

44

¢4

c4

No hemolysis.
Complete hemolysis.

~cConsiderable hemolysis.
g4

44

~4

44

gg

~4

~4

g4

gg

~g

L¢

L4

lg

g~

44

~4

~c

~4

~g

¢4

~4

~4

44

4~

~4

~4

lg

~4

lg

~4

44

4~

~4

lg

L~

* Each tube contained 1 per cent washed human corpuscle suspension 1 cc.

plement in the serodiagnos~s of syphilis. Twenty sera Were tested
for this purpose. All except three of them (Nos. 2, 8, and 9, Table
III) contained sufficient complement in 0.05 to 0.1 cc. to cause complete hemolysis in the presence of 0.01 cc. of the anti-human amboceptor No. 633. No hemolysis occurred, however, in any tube containing 0.005 cc. of the amboceptor and 0.05 cc. of human serum.

HIDEYO NOGUCH1

49

There was considerable, and in some cases complete hemolysis in the
tubes containing the same amount of the amboceptor but 0.1 cc. of
the human serum.
For comparison these twenty sera were also tested upon sheep corpuscles (1 cc. of a 1 per cent suspension) for their natural anti-sheep
hemolysin. The results obtained show that 0.05 cc. and 0.1 cc. both
dissolved the sheep corpuscles in all except the three specimens (Nos.
2, 8, and 9) which failed to dissolve the human corpuscles in the presence of anti-human amboceptor. This means that there were atleast three sera out of twenty which did not contain sufficientcomplement.
Out of 1,250 specimens of fresh human sera so far examined, 1,157
contained enough complement to produce complete hemolysis of
1 cc. of a 1 per cent suspension of human corpuscles in a dose of 0.1 cc.,with the addition of 0.01 cc. of anti-human amboceptor (one unit),
within a period of 20 to 30 minutes at 37°C. in a water bath thermostat. 72 specimens caused a partial hemolysis, and 21 nohemolysis.
Additional amboceptor in doses of from one-half to one ordinary
minimal hemolytic unit to the partially hemolyzed tubes produced
complete hemolysis on a further incubation of 15 minutes or longer.
In the case of the sera, however, which showed no complementary action in the first combination, the addition of an extra quantity of
the amboceptor caused only a tardy partial hemoiysis or no hemolysis.
From the serological standpoint we encounter at least three groups ofhuman sera, those which contain the average amount of complement,
those which contain a subnormal quantity (hypocomplementosis),and those which contain no complement (acomplementosis). Occasionally one meets with a fourth group in which the activity of thecomplement is unusually strong (hypercomplementosis). It is highly
important to take these facts into account in testing human seraby the Bordet-Gengou reaction or the Wassermann reaction insyphilis.
The next step was devised in order to determine whether the insufficiency of complement could not be supplemented b y the additionof an adequate quantity of fresh active serum. That this is easily
accomplished was soon proved.

50

SERUM DIAGNOSIS OF SYPHILIS

Mode of Utilization of Human Complement for the Serodiagnosis of
Syphilis.
The experiments discussed above made it evident that in the majority of fresh human sera there is sufficient complement to cause acomplete and prompt hemolysis of human corpuscles in the presenceof an adequate quantity of the specific anti-human hemolytic amboceptor. Moreover, in cases where there is not enough complement,
an active human serum may be added as a supplement. In fact,
we are now in a position to produce complete hemolysis, and fromthis point it is only another step to test the presence or absence of acomplement-fixing principle in a given specimen of human serum.
One merely measures out a definite amount of the suspected serum
into two tubes and then adds to one of the two an adequate amountof the antigen suspension. Both tubes are incubated for 30 minutes
at 37°C. in a water bath; then the human corpuscular suspension
and the anti-human hemolytic amboceptor are introduced into bothtubes and the contents are well mixed by shaking. The tubes areonce more incubated for 30 minutes at 37°C., and then after another30 minutes or so at room temperature the result is read. It is necessary to shake the tubes two or three times during the incubation.
No result should be taken as final unless the control tube without the
antigen shows complete hemolysis. If hemolysis is incomplete at
the end of the period indicated, adequate modification, which will
be described later, must be made.

Procedure for the Examination of Human Sera Not More than 48 Hours
Old.
I t must be made clear in the beginning that specimens should be
examined as soon as practicable, preferably within 24 hours after
they are withdrawn from t h e patients. After 48 hours, even when
the specimens are kept in the refrigerator, the complement graduallydisappears from the serum. Sera kept for more than 3 days in arefrigerator must be tested by a special technique, to be given later.
Specimens tinged deeply with hemoglobin give unsatisfactory results
and should be rejected. Table IV indicates the amounts of reagentsto be used in the test for fresh human sera and other details regarding
it. The entire set of tubes should be duplicated for each specimen.

S1

HIDEYO NOGUCHIC~

c~

R'~..~

~o~

x--W

0

~

0

"

~q

-I,

v

52

SERUM DIAGNOSIS OF SYPHILIS

Positive and Negative Controls.
Ag in any other serodiagnostic procedure, each serum tested mustbe .acdompanied b y a positive and a negative serum in order to control the reliability of the reagents. In a well appointed laboratory, where many tests are being made daily or every other day,the necessary positive and negative sera will be furnished b y thetests of the previous occasion.

Varieties of Irregular Reactions and Their Adjustment.
Reference has already been made to the possible deficiency of complement in certain specimens of fresh human sera. With these hypocomplementary sera hemolysis in the control tubes without antigen
proceeds slowly and remains incomplete at the end of the second incubation. To these sets of tubes, both the determinative and the control, an additional hemolytic amboceptor unit is introduced in orderto reinforce the hemolytic activity of the complement. In cases inwhich hemolysis is marked only one-half the unit is added, b u t whenhemolysis is slight a whole unit of the amboceptor is needed.
The tubes.should then be put in another rack and subjected to further
incubation until complete hemolysis occurs in the control tubes(without the antigen). 15 minutes or longer may be required.
Specimens which fail to hemolyze' or in which hemolysis is incomplete,even with the additional amboceptor should be tested again by adding
to them a quantity of fresh serum which has been shown to contain an
average complement and at the same time to be devoid of any syphilitic fixation substance (negative serum). In these cases 0.1 cc. of
the complementary serum is used with 0.2 cc. of the acomplementary
serum. The mixture is then tested in the same way that any fresh
serum is tested.

Procedure for the Examination of Human Serum More than 48 Hours
Old.
As previously stated, specimens of human serum which have stoodin a refrigerator longer than 48 hours are inconstant in their complementary activity, and many are markedly deficient. As a rule

~mEYo NOGUCm

53

sera not more than 72 hours old which have been kept constantly inthe refrigerator at 4-6°C. still contain enough complement to makethe test possible. It is best, however, to inactivate all sera whose
complementary activity is no longer a certain factor, supplementing
them with active human complement from negative fresh sera. If
inactivation is complete (55°C. for 30 minutes), the sera are rendered
free of the remnant of their native complement, and what is added
later is of known and uniform quantity. 0.1 cc. of the complement
serum (previously tested) is added to 0.2 cc. of the inactivated serum
and the mixture then tested like any fresh serum. Table V gives thedetails of the procedure.

Procedure for the Examination of Cerebrospinal Fluids.
This procedure is comparatively easy and gives an entirely satisfactory result. It differs from that used for inactivated sera only in
one respect, that of the quantity of the specimen used, which m a yvary from 0.2 to 0.5 cc. No inactivation is required, as the cerebrospinal fluid contains no complement, and 0.1 cc. of active negative
human serum (previously tested) is added as complement (Table VI).

A Method of Preserving the Complement of Fresh Human Serum by
Means of Sodium Acetate.
Rhamy 3 first observed that guinea pig complement remains active
for a long time when mixed with sodium acetate in a strength of approximately 6 per cent of the acetate in the mixture. He recommends
mixing 4 parts of the complement with 6 parts of a 0.9 per cent sodium
chloride solution containing 10 per cent sodium acetate.
Human complement can also be kept active for some time by adding
sodium acetate in a similar proportion. Specimens of human serum
which have been mixed with the acetate can be satisfactorily tested
after several days without the aid of complement from another source,
since the complement remains active for at least 4 days at room temperature (18°C.). Therefore any serum which cannot be examinedwithin 48 hours may be mixed with the acetate saline solution while
perfectly fresh. If a good refrigerator is not accessible the specimen

54

SER~

DIAGNOSISOF SYPHILIS

E_-~

8

8

N

<

gl
[-t

.s~
-~.~ g
~.~

°~

a

4

~ ~.~.~°~,,...,

d

~d

,.-M

~

r...)

.~-~ s@HIDEYO

55

NOGUCHI

o

.m

.~

O9

<

o

~

~

o

56

SERUM

DIAGNOSISO F SYPHILIS

may always be acetated, since the presence of the acetate does notinterfere with the test (Rhamy).
The acetate may be used as preservative for the serum in eitherof two ways. A sterile solution, containing 0.9 per cent sodium
chloride and 10 per cent sodium acetate, may be mixed in equalparts with the serum after separation from the clot; or 0.5 cc. of asterile concentrated acetate solution (50 per cent) in 0.9 per centsaline solution may be placed in a graduated centrifuge tube and theblood drawn from the patient directly into the tube up to the 5 cc.
mark, the whole being then shaken thoroughly. Coagulation takes
place as usual, and the clot is just as firm as in a control tube inwhich 0.5 cc. of saline solution is added to the same amount of blood.
There is no disturbing effect from the presence of the acetate in theserum, either when it is fresh, or when it has stood for several days.
The serum acetate mixture may be kept in the refrigerator, where it
will retain complement activity longer than at room temperature;in the latter, activity is preserved for several days. Once the acetate
serum has become inactive by long standing, it can still be tested
by adding fresh human complement.
The method of mixing clear serum with the saline acetate solution ismuch to be preferred to the direct mixing of the blood and concentrated acetate solution.

Preparation of Reagents.
Although the details with regard to the reagents used in conducting
the present test are essentially the same as those described in earlier
publications 6, 1Gdealing with the anti-human heterohemolytic system(the use of guinea pig complement with anti-human amboceptor andhuman corpuscles), it nevertheless seems desirable to summarize
them briefly here. Antigen, anti-human hemolytic amboceptor, anda suspension of human blood corpuscles are the reagents required totest the patient's serum.

Antigen.--The preparation, titration for its antigenic properties, and preservation of the antigen are described in other publications dealing with the subject
in minute detail,e Sufficeit to say that the acetone-insoluble fraction of tissue
1GNoguchi and Bronfenbrenner, Biochemical studies on so-called syphilis antigen, J. Exp. Med., 1911, xiii, 43.

HmEYO NOGUCHI

57

lipoids is recommended, 0.1 cc. of a 1:10 emulsion with 3 per cent methyl alcohol
solution (stock) and 0.9 per cent saline solution being used for each test. No
preparation which hemolyzes in 0.4 cc. or interferes with complement in thesame dose should be used. The antigen should be effective in doses of 0.02 cc.
of the emulsion. I n the actual test at least five antigenic units are employed inorder that no positive reaction may be overlooked. The methyl alcohol stock
solution remains active indefinitely at room temperature, and a saline emulsionof 1:10 strength may be made up at any time. The latter, when kept in a refrigerator, remains unchanged for several days, although it is best to make a
fresh emulsion on the day that tests are to be made.
Amboceptor.--The production of the anti-human amboceptor is one of the mostimportant parts of the present method. Rabbits, when immunized with thoroughly washed human corpuscles in sufficiently large quantities, yield a powerful
serum, which is able to produce complete hemolysis of 1 cc. of a 1 per cent suspension of human corpuscles in doses of from 0.01 to 0.005 cc. This point will be
discussed at greater length later. The usual amount of this reagent used in thetest is 0.1 cc., which is so made as to represent one hemolytic unit in the presence of 0.1 cc. of fresh human serum and against 1 cc. of the 1 per cent corpuscular suspension, hemolysis becoming complete within 20 to 30 minutes at 37°C.
in a water bath or I hour in an air thermostat.
Corpuscular Suspension.--O.l cc. of a 10 per cent suspension is best suited forthe purposes of the test. The suspension is prepared as follows: 1 part of thewashed corpuscles from any individual (it is convenient to use corpuscles obtained
from one of the patients being bled for test serum) is mixed with 9 parts of 0.0
per cent saline solution. A few cubic centimeters of blood are drawn into a centrifuge tube containing an equal volume of sodium citrate solution (2 per centin 0.9 per cent saline solution) and the nfixture is repeatedly centrifuged withchanges of saline solution until there is no more serum in the supernatant fluid.
The final sediment of corpuscles is suspended in a quantity of saline solution equal
to the original quantity of the blood. The corpuscular suspension thus preparedshould be used when fresh and when not in use should be kept in a refrigerator,
where it can be preserved for a period of about 72 hours. A suspension
older than 72 hours should not be used, however, because the use of corpuscles
with diminished resistance to hemolysis may cause the masking of a possible
weak positive reaction.
Utilization of the Patient's Corpuscles.--For any well equipped laboratory the
preparation of a corpuscular suspension of a definite concentration by the above
method offers no difficulty. There may be occasions, however, for example onboard ship or in field hospitals, when no centrifuge is available. Under these
conditions the preparation of a uniform corpuscular suspension for an entire setof tests is not possible, and to meet this sort of emergency the utilization of thepatient's own corpuscles for testing his serum is recommended. As has alreadybeen emphasized, the serum of the patient to be examined must be perfectly
fresh and the corpuscles which can be liberated by gently stirring the clot are
correspondingly fresh and can be used as the indicator of hemolysis. The fol-

58

SERUM DIAGNOSIS OF SYPHILIS

lowing technique gives the best result: 0.4 cc. of the fresh clear serum from thetube containing the coagulated blood is measured out and put into one of thetwo tubes used for the test. 2 cc. of 0.9 per cent saline solution are added,
making a total volume of 2.4 cc. After putting the remaining clear serum into a
tube for future use, the clot is gently shaken, by means of a medium sized pipette,
to liberate enough corpuscles to tinge the serum dilution to the desired color
value (suspension). The standard of suspension aimed at is 1 per cent of corpuscles, and after a certain amount of practice no difficulty is experienced in
detecting the difference between a 1 per cent and a 1.5 per cent suspension by
this means. A 1 per cent suspension is an opaque fluid with a yellowish red
hue. As the concentration of the suspension increases, the red becomes more
predominant over the yellow tint. Having made a mixture of 0.4 cc. of the
fresh serum and approximately a 1 per cent corpuscular suspension in a total of
2.4 cc., one divides this quantity into two equal portions by measuring out 1.2
cc. into the second tube of the set. One of the two tubes receives the antigen
0.1 cc., and the other receives the saline solution 0.1 cc., and serves as the control without the antigen. Both are incubated for 30 minutes in a water bath or
1 hour in an air incubator. 0.1 cc. of the anti-human amboceptor, representing one hemolytic unit, is then added to both tubes, and after thorough mixing
the tubes are again incubated, as in all other procedures.
When the test is done in this way, the corpuscles are, of course, present
from the beginning, instead of being added after the first incubation simultaneously with the amboceptor, as in procedures in which corpuscles fromother patients are used. But in any procedure the corpuscles may be introduced from the beginning and the final result remain the same.
T s c h e r n o g u b o w s once p r o p o s e d the use o f a suspension of the p a t i e n t ' s blood d i r e c t l y diluted in saline solution in a ratio of 1 d r o p to
1 cc. of saline. T h i s was to serve as the source of c o m p l e m e n t , corpuscles, and, if present, the fixing substance. B y careful scrutiny,
however, one soon discovers t h e e n o r m o u s d i s p r o p o r t i o n a m o n g thevarious elements concerned. F o r example, the a m o u n t of s e r u m p r o b a b l y p r e s e n t in 1 d r o p of blood, w h i c h would p e r h a p s be 0.07 cc.,
would be a p p r o x i m a t e l y 0.035 cc. a t m o s t ( a b o u t one-sixth the a m o u n t
used in t h e w r i t e r ' s system), while t h e c o r p u s c u l a r suspension app r o a c h e s 7 per cent (seven times the c o n c e n t r a t i o n in t h e w r i t e r ' s
s y s t e m ) . T h e a m o u n t of syphilitic antibodies p r e s e n t w o u l d be too
small to m a k e possible the d e t e c t i o n of a w e a k positive reaction,a n d the m i n u t e n e s s of c o m p l e m e n t p r e s e n t in such a m i x t u r e precludes a n y p r a c t i c a l possibility. E v e n a n e n o r m o u s a m o u n t of thea m b o c e p t o r fails to c o m p l e t e hemolysis of such a c o n c e n t r a t e dHIDEYO NOGUCHI

59

suspension of the corpuscles. Moreover, the mixture forms a loose
gelatinous fibrin, involving the whole contents, making it impossible
to stir by shaking. When the first fibrin is removed, a second may
form on further standing. Such a method cannot be used, andTschernogubow himself soon abandoned it.
Emery 9 employs active serum with the cholesterolized alcoholic
extract of heart muscle, disregarding the possibility of obtaining a
false positive fixation with certain non-syphilitic sera. The amountof the patient's serum is minute, but the concentration of the corpuscles (20 per cent of the firmly packed sediment after centrifugation) is almost six times that used in the method being proposed.
There is no economic gain in Emery's method, therefore, since theamount of amboceptor required is no less than that used in the
proposed method. Moreover, the manipulation of minute quantities of various factors by Wright's capillary technique requires aconsiderable degree of skill as compared with the ease with which
regular graduated pipettes can be handled.
Butler and Landon ~° and Myer n inactivate the patient's serum
before the test and add fresh negative human serum from a nonsyphilitic individual as compiement. They recommend the use of
sensitized human corpuscles and the acetone-insoluble tissue lipoids(Noguchi). Their method is decidedly more rational than Emery's
and seems to have given satisfactory results in 300 cases so far reportedJ ~ They also employ Wright's capillary technique.
Thompson ~2uses fresh human serum, with preliminary titration ofeach specimen for its complement activity. This procedure seems to
be unessential, since the majority of specimens already contain enoughcomplement. The amount of corpuscles used is 0.2 cc. of a 2 per centsuspension. A stronger concentration would give a more distinct
reaction.

Results of Practical Application of the Test.
The writer has been able, up to the present time, to examine 1,331
specimens of blood and 52 cerebrospinal fluidsY Of 1,118 specimensof sera from these sources 517 gave a positive and 601 a negative
L71 wish to express my appreciation to the members of the staffs of several
hospitals through whose cooperation this work was made possible.

60

SERUM[ DIAGNOSIS OF S~YPHILIS

reaction, the resltlts conforming to those reported by the serologicaldepartments of the various hospitals. Of 132 specimens from psychiatric cases, 54 were from general paralysis cases, and all except 2
gave a strongly positive reaction. Among other psychoses, including
75 cases of dementia pra~cox, 3 of alcoholic psychosis, 3 of imbecility,
3 of senile psychosis, 6 of arteriosclerosis, 1 of manic-depressive insanity, and 1 paranoic condition, there were only 2 positive reactions,these occurring among the dementia pra~cox cases. The reactions
with 81 inactivated sera agreed with those obtained by others with thesame material. 20 cerebrospinal fluids from cases of general paralysis
gave a strongly positive reaction, while 32 specimens from othernon-syphilitic cases showed a negative reaction. The gtatement will
perhaps bear repeating that of 1,250 fresh human sera complement was
deficient in 93 specimens, which had to be examined either by meansof additional amboceptor or by supplying active human complementfrom fresh negative sera. This special adjustment with fresh sera is
one which demands particular attention on the part of serologists
adopting the method.
Quantitative Consideration of the Complement Fixation Test.
In an ideal method for the serum diagnosis of syphilis every ingredient should be separately controllable by accurate titration. This ispossible with the anti-human heterohemolytic system (Noguchi),in which the amount of guinea pig complement is accurately measured
and added to a definite quantity of serum, whose native complement
plays no part in the reaction, or has been removed by inactivation,and in which the antigen, anti-human amboceptor, and corpuscles areequally definite, no factor being present which can give rise to a quantitative disturbance. The homohemolytic system, upon criticalexamination, will be seen also to be capable of equal accuracy. The
possible sources of error and the methods of adjusting them are discussed below.
The Frequency and Extent to Which a Positive Reaction May Be
Masked by an Excess of Complement.--Only 2 per cent of several
hundred specimens examined in this study were found to contain an
extra unit of complement activity. None contained three units.

HIDEYONOGUCHI61

An excess of complementary activity may cause a positive serum togive a weakly positive or even a negative reaction, but only when theso called antibody content of the serum is less than one fixing unit.
For example, a specimen containing half of an antibody unit in thepresence of two complement units may give a negative result. Nochange in the reaction can occur, however, from an excess of less than
two complement units ir~ the presence of 1½ units of syphilitic antibody. Error from this source is therefore extremely rare; and thereaction cannot be completely negative when the specimen contains
more than one antibody unit.
TABLE

VII.

Relation b'etween Various Quantities of Human Complement and of Antibody.
Syphilitic serum (active).

Amount.

NO. O[
units of

Fresh negative h u m a n s e r u m a s c o m p l e m e n t .

0.1 cc.

0.125 cc.

0.15 c c .

0.2 c c .

0.25 cc.

0.3 C¢.

0.4 c e .

antibody.
i

C6.

0.008
0.016
0.024
0.032

0.5
1.0
1.5

0.04
0.048

2.5
~.0
4.0

0.064

2.0

+++
++++
++++
++++
++++
++++
++++

+++
++++
++++
++++
++++
++++
++++

++
++
++++ ++++++±+ + + + +++++ ++++++++ ++++++++ ++++++++ ++++++++ ++++++++ :+++ ++++++ ++++ ÷++++++ ++++ ++++++++ ++++ ++++
+ + + + ++++ + + + ++Table VII records experiments in which the relation between various
quantities of human complement and of antibody was determined.
The amount of anti-human amboceptor used was that which produced
complete hemolysis of 1 cc. of a 1 per cent suspension of human corpuscles in tile presence of 0.1 cc. of fresh human serum (one complement unit) within 20to 30 minutes at 37°C. (water bath). Table VIIshows that the addition of quantities of the human complement ranging from 0.1 to 0.2 cc. did not materially change the + + + + reaction
when combined with 0.016 cc. of syphilitic serum. The reactions
became somewhat weaker, however, when 0.15 and 0.2 cc. of the complement were used with 0.008 cc. of the serum. When the amountof the syphilitic serum was increased to 0.024 to 0.032 cc. or more,
the reaction was + + + + against 0.3 cc. of the complement, and62

SERUM DIAGNOSIS OF S Y P H I L I S

0.04 cc. of serum prevented hemolysis in the presence of 0.4 cc. ofcomplement. The reduction in the degree of positive reaction
caused by the variations of 0.1 to 0.2 cc. of the human complement is
insignificant and has no serious effect upon the ultimate result when
the serum contains more than 1½ antibody units. The apparent disproportion of complement in the routine amount of 0.2 cc. of the
fresh serum is well balanced by the antibody content, which is four
to five times that of the same serum inactivated. For that reason
only 0.1 cc. of human serum is added to 0.2 cc. of inactivated serum.
This proportion is similar to that of the guinea pig complement andinactivated serum used in the Wassermann system and in the antihuman heterocomplement system (Noguchi). But the addition of
0.2 cc. of the human complement does not mask a strongly positive
reaction ( + + + + ) .

The Possibility of Interference by Negative Serum in Complement
Fixation in the Itomohemolytic System.--The use in the homohemolytic system of fresh negative serum as complement when there is nocomplement or too little in the specimen to be examined, while entirely
analogous to the use of fresh guinea pig serum as complement for an
inactivated patient's serum, may rouse apprehension as to the possibility of reduction in the degree of fixation by this comparatively
large amount of human complement, owing to its indifferent serum
constituents. The experiments recorded in Table viii, in whichhuman and guinea pig complement were studied in parall61 series,
show that the addition of an inactivated negative serum to a syphilitic
serum does not cause any so called complementoid blocking of thecomplement fixation of any significance, and that no error can result
from this source.
All the other experiments of this sort with syphilitic sera gave similar results. It was found, however, that a syphilitic serum containing
less than one-half an antibody unit caused less inhibition in the tubes
to which more than 0.3 cc. of the inactivated negative human serum
had been added, but never completely masked the reaction. There
was no appreciable difference in the tubes containing 0.1 or 0.2 cc. of
the inactivated serum and that containing none. The addition,
however, of inactivated guinea pig serum (56°C.) to a syphilitic serum
produces marked weakening of the fixation reaction, and even corn-

HIDEYO

63

NOGUCHITABLE VIII.
Effect of Inactivated Negative H u m a n Serum upon the Complement Fixation.
With human complement.

Results.

With guinea pig
Acmpl . . . . t.

Results.

With inacti- Syphilitic
serum
vated
(56°C.)
syphilitic
containing
serum.
1 antibody
unit
0.2 cc.
Humancomplement (active
serum)
0. l "The same 4negative
human
serum
(56°C.)
0.1 "The same 4negative
human
serum
(56°C.)
0.2 "The same +negative
human
serum
56°C.)
0.3 "Fhe same 4negative
human
serum
(56°C.)
0.4 "Allgave [Syphilitic
completeserum(56oc.)
fixation,
regardless containingof the ad1 antibody
dition ofunit
0.2 cc. /the inac- Guinea pig
tivatedcomplement 40
negative
serum.
per cent 0.1 " 1

With active Active syphilitic sesyphilitic
rum, conserum.
taining 4
antibody
units
0.2 cc.
The same 4negative
humanCompletefixati~in all.

serum(56°C.)

0.1 "JThe same 4negative
human
serum

(56°C.)
0.1 "The same +negative
human
serum

(56°C.)
0.2 "The same 4negative
human
serum
(s6oc.) 0.3 "The same 4negative
human
serum
0.4
"
(56°C.)

All
gave
completefixation,no interference
being observed
from theaddition of
the inactivatednegative
human
serum.

64

SERUM DIAGNOSISOFSYPHILISTABLE VIII--Concluded.
With human complement.

With active The same +syphilitic
negative
serum.
human

Results.

With guinea pig
complement.

Results.

Completefixationin all.

serum(56°C.)
0.2 cc.
The same +negative
human
serum
(56°C.)
0.3 "The same +negative
human
serum
(56°C.)
0.4 "

plete blocking when more t h a n 0.3 cc. of t h a t serum is added to one
syphilitic a n t i b o d y unit. This confirms earlier observations, isResult of the Presence of an Excess of Amboceptor in the Complement
Fixation T e s t . - - T h e disturbance resulting from an excess of a m b o ceptor in a n y c o m p l e m e n t fixation test has been r e p e a t e d l y pointed
out and is recognized b y i m p a r t i a l workers as inherent in the antisheep hemolytic system. R e m o v a l b y absorption of the n a t u r a lanti-sheep a m b o c e p t o r from each specimen of serum prior to test has
been advocated, b u t for obvious reasons is impracticable when several
dozen specimens m u s t be examined at one time.
I n T a b l e I X are recorded the results obtained in parallel series oftests, 19 the anti-sheep s y s t e m with guinea pig c o m p l e m o n t being usedin one series, and the a n t i - h u m a n homocomplernent system in theother. These experiments confirm earlier observations 2° t h a t a
is Noguchi and Bronfenbrenner, The interference of inactive serum and eggwhite in the phenomenon of complement fixation, J. Exp. Med., 1911, xiii, 92.
19These' tests were carried out by Major Felix R. Hill, Captain George L.
Schadt, and Lieutenant Ralph R. Simmons in this laboratory.
2oNoguchi, Die quantitative Seite der Serodiagnostik der Syphilis, Z. Immunitgtsforsch., Orig., 1911, ix, 715.

HIDEYO

65

NOGUCHIpositive serum containing 1 a n t i b o d y unit can become completely
negative when 4 amboceptor units are used, or 3 a n t i b o d y units with
20 a m b o c e p t o r units. Table I X shows t h a t 1 a n t i b o d y unit is m a d enegative b y 6 a m b o c e p t o r units and 3 a n t i b o d y units b y 10 a m b o c e p t o runits with the Wassermann system, while with the a n t i - h u m a n
homocomplement system nearly twice the a m o u n t of a m b o c e p t o r isrequired to produce the same effect.
T h e p h e n o m e n o n just described assumes practical importance ina n y system in which are used foreign blood corpuscles for which h u m a nserum normally contains varying amounts of. natural hemolytic
amboceptor, in which case there is a possibility of an excess of amboceptor in the test. I t does not occur in an anti-human hemolytic
T A B L E IX.

Reversion of Reaction through Excess of Amboceptor.
Syphilitic antibody 1 unit.
No. of unitsofWass~mann
Anti~umn
amboceptor, anti-sheepbomocomplesystem,
ment system.

++++
++++
++

1

2
3
6
10
20
40

-

++++
++÷+
++÷+
+++

Syphilitic antibody 3 units.

Syphilitic antibody 10 units.

W~sermann
antPsheep
system,Anti-humanhomocompl~
ment system.

Wassermann
anti-sheep
system,Anti-humanhomocomplement system.

++++
++++
+++
+

++++
++++
++++
++++
++

++++
÷+++
++++
++++
++++
+++
+

++++
++++
++++
+÷++
++++
++÷
+

-

TABLE

X.

Titration of Natural Anti-Sheep Amboceptor in Human Serum.
No. of units o~ anti-sheep amboceptor in 0.2 cc. of
human serum (56°C.), titrated with g u i n e a
pig complement.

ro. of
peel-

Sera.

i

hens
xamned.

5

6

7

8 >10

- -

Syphilitic sera . . . . . . . . . . . . . . . . . . .
Non-syphilitic sera. . . . . . . . . . . . . . . . .
Normal sera . . . . . . . . . . . . . . . . . . . . . .

190 19 21/ 42 21 25[ 26 17 8 41 3[ 4111
151 25 3~ lo 112 i 8 525
2 2 3:1

Total . . . . . . . . . . . . . . . . . . . . . . . . . . . .

326 ,~3J

56 38 391 27 15

66

SERUM DIAGNOSIS O F

SYPI-IILIS

system, whether guinea pig complement or human complement isused, and especially when the patient's own corpuscles are employed
for each specimen. Data which have been collected concerning theamount of natural anti-sheep amboceptor in 326 specimens of humanserum are given in Table X.
Tables I X and X explain the possibility of a reduced or reversed
reaction with certain syphilitic sera in the anti-sheep system. The
frequency and extent of error from this source are much greater thanthose due to slight variations in complement content, which are readily amenable to quantitative adjustment.

Quantitative Estimation of the So Called Syphilitic Antibody.--If
necessary, any strongly positive serum can be titrated by the homocomplement system, the procedure being the same as that recommended for the anti-human heterocomplement system; namely, thatof keeping all the other ingredients constant and determining thesmallest quantity of the serum which will give complete fixation.
For this purpose the serum may be inactivated before titration. To
a number of tubes containing 0.1 cc. of the fresh human complement(previously titrated) and the standard antigen (usually cpntained in
0.1 cc. of a suitable dilution) are added varying amounts (ranging from0.01 to 0.2 cc.) of the positive human serum (inactivated) to betitrated, and the mixture is made up to 1.3 cc. in each tube. Afteran incubation at 37°C. for 30 minutes in the water bath or 1 hour in an
air incubator, the corpuscle suspension (0.1 cc.) and one unit ofamboceptor (0.1 cc.) are added, and a second incubation follows.
The smallest quantity of serum which produces complete fixation istaken as one antibody unit.
SUMMARY.

The elimination of the foreign complement and corpuscles from thetest for the serodiagnosis of syphilis has been attempted, and the results so far obtained are very satisfactory. Instead of using guinea
pig complement, fresh human serum was utilized for the source ofcomplement for the production of hemolysis upon the human corpuscles in the presence of an adequate amount of the specific antihuman amboceptor (prepared in rabbits). Usually 0.1 cc. of fresh

HIDEYO NOGUCHI

67

human serum contains enough complement to hemolyze 1 cc. of a 1
per cent suspension of human corpuscles, but the amount of antihuman amboceptor required in this combination isabout five to seventimes that necessary when guinea pig complement (0.04 cc.) is used.
It has also been shown that when a given human serum contains insufficient complement, an adequate quantity (0.1 cc. is usually
enough) of another fresh negative serum may be added to supplement
it. However, one rarely encounters this group of sera. Inactivatedhuman sera can also be examined by utilizing human complementfrom another source (the serum must be fresh, active, and negative).
The only drawback to the present method is the comparatively large
amount of anti-human hemolytic amboceptor required. It is estimated that 30 to 40 cc. of the anti-human hemolytic immune serum,
from one rabbit, of high potency--say 0.005 cc.--would be enough to
examine about 3,000 to 4,000 cases (0.01 cc. for each case), whereas if
guinea pig complement were used the same amount would cover about
15,000 tests (0.002 cc. for each case). In a large hospital or in the
Army there should be no difficulty in preparing any amount of theanti-human hemolytic amboceptor. For example, material for 100,000
tests could be prepared within 1 month in less than 100 rabbits. The
amboceptor serum can be used in the fluid state, or, if the titer ishigh, impregnated into filter papers.
Special attention should be called to the fact that to obtain a powerful anti-human hemolytic amboceptor five to six intraperitoneal injections of corpuscles, thoroughly washed (until there is no trace ofserhm in the supernatant fluid), in doses of 5, 7, 10, 10, 10, and possibly another 10 cc. of the concentrated suspension (restored to theoriginal volume of the blood) are required. 21 The bleeding may bedone by the 9th or 10th day. The animals may be kept after
bleeding for the production of more amboceptor by subsequent injections of the washed corpuscles.
Finally, it should be emphasized that only the acetone-insoluble
fraction of tissue lipoids of required standards (Noguchi) should be
used when utilizing the human complement in the fixation test.
21Intravenous injections of 3, 3, 4, and 4 cc. of the corpuscular suspension
every 4 or 5 days also give excellent results.