野口英世著

Journal of Experimental Medicine

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I N F L U E N C E OF T E M P E R A T U R E UPON T H E VELOCITY
OF T H E C O M P L E M E N T F I X A T I O N R E A C T I O N
IN SYPHILIS.
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rocke[eller Institute for Medical Research.)(Received for publication, April 25, 1918.)

It has become customary to carry out immunity reactions in vitro
at the temperature of 37°C., because most of the reactions between
specific antibodies and antigens take place only at 37°C. or near thatpoint.. The reactions of bacteriolysis, cytolysis, hemolysis, agglutination, precipitation, phagocytosis, opsonization, etc., manifest theirmaximum activities at 37°C. Certain biological reactions, however,
do not necessarily follow this general rule. For example, hemolysis
produced by saponin, bile salts, cobra lecithid, or sodium oleate iscomplete within a very short time whether at 4° or 37°C? ,2 The
velocity of the reaction in these instances is such that time and temperature play but a slight part.
The mechanism of the lipotropic complement fixation of syphilitic
serum or spinal fluid is not understood, but it is certain that thelipoids are an important factor. The question in regard to thevelocity of this reaction has not received as much attention as it deserves. The prevailing idea is that it is one of the immunity reactions which manifest their maximum activity at 37°C. Whether or
not the reaction can take place at lower temperatures has not been
carefully studied. We have been interested in this phase of theproblem and have carried out numerous experiments to determine therelation between time, temperature, and reaction. The determination
1Madsen, T., and Walburn, L., Toxines et antitoxines. L'influence de latemp6rature sur la vitesse de r~action. I, Overs. 1,,.Danske Vidensk. Selsk. Forh.,
1904, No. 6, 425.
2 Madsen, T., and Noguchi, H., Toxines et antitoxines. L'influence de la ternp6rature sur la vitesse de r~action. II, Overs. k. Danske Vidensk. Selsk. Forh.,
1904, No. 6,447.
297

298

COMPLEMENT F I X A T I O N REACTION I N SYPHILISof these points is of practical importance at the present time, since,should the reaction prove to take place satisfactorily at a temperature
which can be obtained without the aid of a special incubator, the performance of the test becomes much more widely adaptable. That thisis the case is shown in the following experiments.

Detection of Varying Known Quantities of the Fixing Substance (" Syphilitic Antibody") at Different Temperatures.
It was the purpose in this series of experiments to study whether or
not a given quantity of the fixing substance can be detected, not onlyat the usual incubation temperature of 37°C., but also at 30 ° or 23 °.
Graduated doses of a strongly positive syphilitic serum were chosen, sothat a series woul.d represent two and one fixing units and threefifths and one-fifth of a unit. For the first experiment a serum wasselected which produced complete fixation in a dose of 0.05 cc. within
30 minutes at 37°C. (water bath). Of this 0.1, 0.05, 0.03, and 0.01 cc.
were measured into different tubes and tested for their fixing capacity
at different temperatures, 37 °, 30 °, and 23°C.
In the first series human complement, 0.1 cc., and in the second
guinea pig complement, 0.04 cc. (or 0.1 cc. of 40 per cent guinea
pig serum dilution), were used. The quantities of the other elements were as usual, antihuman amboceptor, 0.1 cc., containing one
unit for the human and two units for the guinea pig complement,
10 per cent human corpuscle suspension, 0.1 cc., and antigen, 0.1 cc.
The total volume in each tube was made 1.5 cc.
Four groups of tubes, representing 0.1, 0.05, 0.03, and 0.01 cc. ofthe serum, were prepared for studying the influence of temperature
upon the velocity of the reaction (Table I). Each group had seven
duplicate sets for each dose of serum, as it was necessary to add amboceptor and corpuscle suspension at seven different intervals after the
first incubation period began. The addition of the amboceptor andcorpuscle suspension was m a d e simultaneously 10 minutes, 20 minutes, 30 minutes, 60 minutes, 2 hours, and 4 hours after the contents
were mixed.
For the temperature of 37°C. a water bath was used, for 30°C. aspecial thermostat room, and for 23°C. the laboratory room. Not

HIDEYO N O G U C H I

299

only the first incubation, b u t also the second or hemolytic phase of thereaction, was carried out at the temperatures indicated; t h a t is, theresults recorded under the heading of 23°C. were obtained at thattemperature throughout, and those at 30 ° and 37°C. also.
In the experiments recorded in Tables I and I I the same quantitiesof fixing substance (" syphilitic antibody") were employed, b u t thesource and a m o u n t of complement used were different. I t will be
noted t h a t the reaction was somewhat stronger when 0.04 cc. of guinea
TABLE I.
Fixation of Human Complement at Various Temperatures.

37oc.

I

3o.c.

I

23.c.

Serum.

Imme. . . . . .
diately.
l O m i n . + + + + / ÷ + [ -- -- + + +_+20 [ + + + + / + + + l + t-1++++ + +30 " l/++++I++L~:I + + + + + + + / + L-/++++L + + I++++J++l~J + + + + + + + l
60 " I]++++[++[~1++++I++L~/++++L++++I~ ~,,Human complement, 0.1 cc.; diluted antihuman amboceptor, 0.1 cc. (one unit);
10 per cent human corpuscle suspension, 0.1 cc.; antigen, 0.1 cc. Total volume,
1 . 5 cc.
The second incubation also was carried out at the temperatures indicated.
pig complement was used instead of 0.1 cc. of h u m a n complement.
The difference seems to be due to the fact s that the more active thecomplement the easier it is fixed, since guinea pig sertml contains therequisite complement in a much smaller volume.
Some striking facts are brought out in these experiments. W h e n
the a m o u n t of fixing substance exceeds two units (0.1 cc.), completez Noguchi, H., and Bronfenbrenner, J., The interference of inactive serum andegg-white in the phenomenon of complement fixation, J. Exp. Med., 1911, xiii, 92.

300

COMPLEMENTFIXATION

REACTIONIN SYPHILIS

fixation takes place within 20 minutes at .23° or 30°C., while at 37°C.
the fixation is complete within 10 minutes. When one unit of fixing
substance (0.05 cc.) is used, the fixation is complete within 30 minutes
at 37°C., 60 minutes at 30°C., and 2 hours at 23°C. With three-fifths(0.03 cc.) and one-fifth (0.01 cc.) of the fixing unit the minimum timerequired for completion of the fixation is 30 minutes for 37°C., 60
minutes for 30°C., and 2 hours for 23°C.
TABLE II.

Fixation o[ Guinea Pig Complement at Various Temperatures.
37°C"
Incubation
period.

II30°C"Serum.
1I[¢5Immediately.
lOmin.
20 " + + + + + + + + + +30 " + + + + + + + + + +60 " / + + + + l + + + + l + +2 hrs.
4"23°C"¢5¢5_+++% ++;÷+ + + + ÷ .2f- . . . .
-++++~+++++ i - ++++l + +~ + + + + +4++~-+ -+-+ ±~ i++++ ++ l ++++- --+- +++ + + + +4 ~-+++
I + + + + +4~-+ -+ ~:1++++1++--+
D--

40 per cent guinea pig complement, 0.1 cc.; diluted antihuman amboceptor,
0.1 cc. (representing two units for this system) ; 10 per cent human corpuscle suspension, 0.1 cc.; antigen, 0.1 cc. Total volume, 1.5 cc.
The second incubation also was carried out at the temperatures indicated.
The velocity of the reaction of complement fixation and subsequent
hemolysis is much greater at 37°C. than at 30 ° or 23°C. A q u a n t i t yof complement which will completely hemolyze the mixture in 20
minutes at 37°C. will require 35 minutes at 30°C. and 45 minutes
at 23°C. for complete hemolysis. I t is therefore understood t h a t thetest of syphilitic serum at 30 ° or 23°C. requires a period of nearly 3
hours, while at 37°C. the reaction is complete at the end of 1 hour.

301

HIDEYO NOGUCHITABLE HI.

Comparative Study of Specimens Tested at Different Temperatures.
Specim,m.

Serum 1
u

2

"
¢c

3
4

"

5"

6"

8"

9"
"
"
"
"
"
"

10
11
12
13"

14

15
16
17"
18
" 19" 20
Cerebrospinal
fluid 1
Cerebrospinal
fluid 2
Cerebrospinal
fluid 3
Cerebrospinal
fluid 4
Cerebrospinal
fluid 5

37°C.

30°C.

23*C.

Incubation 30 mln.

Incubation 2 hrs.

Incubation 2 hrs.

++++

++++

+++n-++++
++++

++++
++++

++++
++++

+++

+++

+++

++

++

++

~Iilount.

0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.5

++++
+

++++
+

++++

++++

++++

++++

0.5

++++

++++

++++

+++

+++

+

0.5
0.5
0.5w

+++

H u m a n c o m p l e m e n t was employed. D i l u t e d a n t i h u m a n amboceptor, 0.1 cc.
(one u n i t ) ; 10 p e r cent h u m a n corpuscle suspension, 0.1 cc.; antigen, 0.1 cc.
T o t a l volume, 1.5 cc.
T h e first a n d second i n c u b a t i o n s were carried out a t the t e m p e r a t u r e s indicated,

302

COMPLEMENT F I X A T I O N REACTION I N SYPHILIS

Examination of Specimens of Serum and Cerebrospinal Fluid atDifferent Temperatures.
With a view to the possible practical application of the facts justmentioned, a series of tests was undertaken with specimens of serumand cerobrospinal fluid at 37 °, 30°, and 23°C. The serum was usedin the active state, and the cerebrospinal fluids were tested by adding
human complement, 0.1 cc., to each specimen. Table I I I gives theresults.
There was no disagreement in the results obtained at the threedifferent temperatures. Apparently the examination of syphilitic sera
and cerebrospinal fluids can be conducted in a warm laboratory without recourse to an incubator in which a temperature of 37°C. is maintained. It is advisable, however, to make use of the incubatorwhenever it is available, and, when not accessible, to make the roomas warm as possible, in order to accelerate the reaction. 1 hour ofincubation is sufficient when the temperature of the air is 32-37°C.
CONCLUSIONS.

Examination of syphilitic serum or cerebrospinal fluid can be madeat any temperature between 23 ° and 37°C. The velocity of the fixation reaction, including the fixation of complement and subsequent
hemolysis, is greater at a higher temperature, the optimum point
being 37°C. The maximum reaction is also reached, however, whenthe mixture of lipoids, syphilitic serum, and complement is allowed tostand for a long enough period at a lower temperature, the minimum
thermal point being near 23°C. For the optimum temperature (37°C.)
an incubation of 30 minutes is sufficient, while for the minimum temperature (23°C.) 2 hours are necessary. At the temperature of 30°C.
the reaction proceeds with moderate velocity and is complete within
60 minutes.
Guinea pig complement gave a sharper reaction with the sera whichcontained less than one unit of the fixing substance. Fixation iscomplete, however, at any of the three temperatures within 20 minutes
when there are more than two units present. A serum containing one
unit of fixing substance will complete reaction within 30 minutes at

HIDEYO NO GUCI:FI

303

37°C., 60 minutes at 30°C., and 2 hours at 23°C., irrespective ofwhether human or guinea pig complement is used.
For many reasons a properly adjusted thermostat for 37°C. isrecommended for conducting the serum diagnosis of syphilis whenpossible, but it should not be overlooked that at a temperature near30°C. an entirely reliable result can be obtained without a special
incubator. Even at a temperature as low as 23°C. the test can becarried out if sufficient length of time is allowed.
The foregoing conclusions refer only to the systems in which theacetone-insoluble fraction of tissue lipoids 4 is used as antigen.
4 Noguchi, H., Serum diagnosis of syphilis and butyric acid test for syphilis,
Philadelphia, 3rd edition, 1912.