野口英世著

Journal of Experimental Medicine

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V A R I A T I O N S IN T H E C O M P L E M E N T A C T I V I T Y A N D
FIXABILtTY OF GUINEA PIG SERUM3BY H I D E Y O N O G U C H I Am~ 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.)

Some time ago one of us (Noguchi) encountered a peculiar action in goat serum. This serum, while fresh and active, remained
undeviated by the Wassermann reaction or by any combination ofspecific antigen and antibody. It was natural, therefore, to inquire
whether a similar action might not be found in the sera of otherspecies of animals or in the sera of individuals of a species in which
the complement is generally capable of deviation.
Our present paper concerns itself with the quantitative determination of the complementary activity of various sera and their behavior towards the complement deviation phenomenon.
The most important and most widely studied of all complements
is that' of the guinea pig. Although there are some observations
bearing upon the relative complementary activity of different specimens of this sert~m, there is no work in which quantitative estimations of complementary activity of a large number of specimens
with simultaneous determinations on the fixability of each has been
carried out. This has been done in our present studies.
We have also studied the complementary activity of the sera ofseveral other species, such as beef, sheep, goat, pig, dog, cat, rabbit,rat, chicken, etc. 2 Some of these sera were tested for their fixability, our purpose being to see whether the complements of any ofthese could be used for the fixation test.
METHOD OF STUDY.

I. The Titration of the Activity of Comple~nent.--The amboceptor derivedf r o m rabbits immunized with washed h u m a n corpuscles was used in all theReceived for publication, September 2I, 191o.
2 T h e results with these sera will be published separately.

69

Variations in Guinea Pig Serum.

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d~666dddddd~6dd666ddd6~dHide~]o Nogueki and J. Bronfenbrenner.

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72

Variations in Guinea Pig Serum.

hemolytic experiments. I n addition to this, another ambocep~or derived f r o m agoat similarly immunized was employed in certain experiments recorded in thelatter part of this paper. Unless otherwise stated, however, rabbit amboceptor
is r e f e r r e d to.
T h e amboceptor was used in con,stant doses. F o r each iube o.I c.c. o f aIO per cent. suspension of washed h u m a n corpuscles was used. T h e complemen,t to be titrated was added to a series of tubes in graduated quantities. T h etotal volume of fluid in each tube was b r o u g h t up to I c.c. by means of theaddition of o.9 per cent. salt solution. T h e tubes were placed in a water t h e r m o stat a n d incubated for two hours at 37 ° C.
2. The Determination of the Fixability of Cornplement.--In determin~ing thefixation of complement, the system used was the one introduced by Noguchi forthe diagnosis of syphilis. T h e a m o u n t of at~tihuman amboceptor ( r a b b i t ) wasuniformly two units for each tube T h e quantity of washed corpuscles used
was o.I c.c. of a IO per cent. suspension. T h e quantity of complement varied
according to the titers of the different sera, T h e amount of antigen emulsion
was o.i c.c., which contain~ed about four antigenic units. I t was made f r o m theaceton-insoluble fraction of lipoids f r o m a h u m a n liver, according to Noguehi's
met_hod3 I n doses of o.45 c.c. it h a d no anticomplementary action. The serum
was a mixture f r o m several untreated cases of secondary syphilis a n d was inactivated before use. I t h a d r~o inherent anticomplementary property in doses of
0.2 c.e., and in the fixation experiments the quantity employed was only onet e n t h of this.
T h e determination of the amount of fixed complement was made by reading,according to the colorimetric method of Madsen, the degree of hemolysis present in a tube where an incomplete fixation h a d taken~ place, and then calculating
the approximate amount of complement still free. As the exact amount ofcomplement originally .employed is known; it is an easy matter to calculate thea m o u n t of complement which h a d been fixed.
T h e results obtained are given in Table I. I n this series of experiments
forty-one guinea pigs were examined at the same time.

In studying the results summarized in Table I, several interesting
facts may be noticed. It was rather unexpected to find that the complementary activity increased more or less after the sera remainedin contact with the clot for forty-six hours, when compared withthe activity of the same sera left in contact with the clot for twentyhours after the bleeding. A few sera, however, became somewhat
weaker under the same circumstances. The following analysis
gives the results that were obtained.
The average titers for the first (twenty hours) and second(forty-six hours) determinations are nearly the same, being o.o216
cubic centimeter for the first, and o.o2o 9 cubic centimeter for theSerum Diagnosis of Syphilis, ISt edition, Philadelphia, 191o, p. 71.

73

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 e r .

Number ofsera examined.
Determination after ~o hours.
Determination after 46 houra

Comparable titers.

o.o13 c.c.

oI

0.01~ C.C.
o.o17 c.c.

212I

20.0:8 c c.

650.02 C.C,

IO

40.022 c.c.
o.o2~c.c.

:o9:o4o.o27 c.c.

2o0.0 3C,C.

IO0 . 0 3 5 c.c.
0.04 c.c.

ooo20.06c.c.

0I

41

41

second. It will be noticed, also, that the differences between thehighest and the lowest titers are much greater with the second
determination. To insure greater uniformity in action, it is preferable to employ sera which have been left in contact with the clot
for about twenty-four hours.
The titers of these forty-one sera are not absolute; they can bemade higher by employing a larger quantity of amboceptor. W esought not absolute, but relative titers in our present experiments,
and the following chart shows the frequency with which the same
titers were obtained with different sera.
CHARTI.

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JTiters.
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Variations in Guinea Pig Serum.

74

Let us now consider the relative susceptibility of different sera tothe fixing phenomenon in the syphilis reaction. Here we have two
Number ofspecimens
having thetiters indieared,

Amount of guinea
pig serum fixed,

Amount of guinea
pig serum fixed,

Number ofspecimens
having thetit.era indicared.

40.0 ( n o n - f i x a b I e )~o.I2 c.c.

0.03

c.c.

x

o.135 c.c.

i0.0~

C.C.

IO.I 4C.C.

I

0.075 c.c.

I3o . I 4 7 c.c.
o.z5 e.c.

z0.085 c.e.
0.09

c.c.

24

0.24

Io.Io5 c.c.

Izc c.

4I
C H A R T 2.

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Hideyo Noguchi and J. Bronfenbrenner.

75problems. The first is to determine the absolute quantities of different sera fixed by given constant amounts of syphilitic serum andantigen; and the second is to determine the number of units of complement that have disappeared. The absolute amounts of sera fixedunder the same conditions vary consideraMy with different specimens, but the variation in the majority of the specimens is slight.
The contrast between the extremes in the fixation of guinea pig
serum is most striking. The non-fixable serum (No. 34) showed noabnormal complementary activity, and the highest fixable serum(No. 33) had also the usual titer of activity, although the latter deteriorated with an unusual rapidity during forty-six hours. Anotherspecimen (No. 38) had also shown a rapid deterioration, but there
was no abnormality in its fixability.
Taking up the same subject from the standpoint of unity of complementary activity, the following statistics were obtained. Ingrouping the units, the fractions intermediate between two standard
divisions were included in the nearest division. Several arbitrary
zones of fixability may be created (see below) in order to enable usto gain a better idea of the occurrence of various degrees of fixability among the sera thus examined. In the zone of normal fixability
Number of units ofcomplement fixed.

~o.
1.5I}
I

Zone of non-fixability, I specimen (2.4 per cent.).

2.

0

2.5
33.5

2l
4

Zoneof inferior fixability, 8 specimen,s (I9.5 per cent.).

4-5
5.

95

Zoneof normaI fixability,20 specimens (48.7 per cent.).

4- 6}

6.
6.5
5-5
7.
7.5
8.
8.5
9.
9.5

31o
5IIIooI
41

Zone of superfixability,12 specimens (29.2 per cent.).

76

Variations in G*ginea Pig Serum.

are included the specimens which varied from the average by 0. 5 ofa unit in either direction.
C~A~T 3.

~o~ea(1o|•"I%o\J ~

2~

45@

789J

1oY
Units of complement fixed.

The average amount of serum fixed was 0.098 cubic centimeter(see Table I, p. 71) and the average number of units fixed was4.64 (see Table I, p. 7 I ) . In fact, the majority of sera possesseda susceptibility to fixation not far removed from the average.
SU IVIM ARY.

The following conclusions may be drawn from the foregoingseries of experiment s. The comptementai W activity varies withina definite limit in different specimens of guinea pig serum. Withsera which stood in contact with the clot for twenty hours, thestrongest and weakest were in the ratio of o.oi 5 cubic centimeter to0.04 cubic centimeter. The former was 2.66 times stronger than thelatter. The variation observed with the same series of sera after
forty-six hours was still more striking. The strongest was o.oI 3
cubic centimeter, and the weakest, 0.06 cubic centimeter, that is, theformer was 4.6 times stronger than the latter. These findings
agree with those made by Massol and Grysez. 4 The variations* Massol and Grysez, Compt. rend Soc. de biol;, 191o, lxvii, 588.

Hidego Nogttchi a~d J. Bronfenbrenner.

77

were not so marked with the majority of sera. It is noteworthy thata large number of the sera gained in the complementary activity
when remaining in contact with the clot for forty-six hours, whilesome sera became weakened during the same length of time.
The amount of serum fixed by given constant quantities of syphilitic serum and antigen varies much more markedly than the variations in their complementary activity. One serum failed altogether
to be fixed. On the other hand, one sample of serum was so easily
fixable that o.24 cubic centimeter (corresponding to 9.6 complement
units of this specimen) disappeared, while the average quantity
fixed was only o.o98 cubic centimeter (corresponding to 4.64 complement units). The normal standard of fixability was shown in
about 5° per cent. of the specimens examined. If the zone ofnormal fixability is enlarged in both directions by one unit, the percentage of normal fixability would become 65.8. There is no definite relationship between the complementary activity and the fixability of a given specimen of guinea pig serum.
The facts derived from our present experiments, especially in regard to the exceptions in the fixative quality of this serum, demand
the utmost precaution from those intending to employ it for diagnostic purposes, as, for example, in the Wassermann reaction. No
quantitative work is possible with the complement fixation reaction
unless the experimenter is capable of determining the fixability ofthe serum in use. One of us (Noguchi) has long realized thissource of error, and in order to reduce it he has advised the employment of a mixture of sera from more than two guinea pigs.