野口英世著

Journal of Experimental Medicine

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T H E I N T E R F E R E N C E OF I N A C T I V E SERUM AND EGGW H I T E IN T H E P H E N O M E N O N OF COMPLEMENT FIXATION3BY HID,EYO N O G U C H I AND J. B R O N F E N B R E N N E R .

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

Is the fixative action of a specific antigen-antibody combination
directed only towards the serum constituents known as complement
or towards any protein whose physico-chemical properties withregard to the fixation phenomenon are similar to complement?
This question needs experimental determination, as the complementary activity of a given specimen of serum has no constantquantitative relationship to the amount of other non-complementary
protein constituents contained in the same serum.
If the fixation were directed exclusively towards the active complementary constituents, we could measure quantitatively the fixing
capacity of the combination of a given specific antigen and antibody
by simply estimating the units of complement fixed. On the otherhand, if the fixation is also directed towards the other indifferent
proteins simultaneously present in the complement-containing
serum, the quantitative estimation of the fixative capacity of theantigen-antibody combination becomes complicated; we cannot
measure it by the units of complement fixed, for the fixation is also
shared by the other serum components whose exact amounts cannot
be ascertained by this method. A great miscalculation of the real
fixing capacity must inevitably result when one uses as indicator
the complementary activity alone. This source of error is steadily
increased by the gradual disappearance of the complementary
activity from the serum, while the amount of ~he other non-complementary constituents not only remains undiminished, but is increased by the gradual conversion of the active complement into an1Received for publication, September 21, i910.

92Hideyo Noguchi and J. Bronfenbrenner.

93

inactive complementoid. Before this fundamental relation is established, any attempt to estimate complement fixation quantitatively
is premature.
In this communication we present the results which we obtainedin studying the effects of various non-complementary proteins upon
the complement fixation phenomenon, including the syphilis reaction. In our study we employed, as usual, the antlhuman hemolytic system recommended by Noguchi. Every factor concernedin this system is under perfect quantitative control.
THESYPI-IILIS REACTION.

In this series the effects of various inactivated or spontaneously
deteriorated sera and egg-white were studied. Varying quantitiesof each serum and of egg-white, in a twenty per cent. dilution in0. 9 per cent. salt solution, were added under three different conditions to the fixing mixture of syphilitic serum and antigen (acetoninsoluble fraction of Iipoid from human liver). They were added
and incubated ( i ) before the addition of complement; (2) at thesame time as the complement; and (3) after the complement had
been fixed. Some tubes without any serum or egg-white were, ofcourse, provided as controls. The amount of syphilitic serum used
was 0.02 cubic centimeter for each tube, and this amount was capable of fixing 0.08 cubic centimeter of guinea pig complement in thepresence of o.I cubic centimeter (four units) of the antigen emulsion. In the case of non-anticomplementary sera, the amount ofcomplement used was uniformly 0.04 cubic centimeter of fresh
guinea pig serum. On the other hand, certain deteriorated sera
showed a more or less anticomplementary property. This had to beovercome by adding, in addition to the 0.04 cubic centimeter ofcomplement, the exact amount of active complement (guinea pigserum) necessary to neutralize each such specimen. The total
volume in each tube was made equal to one cubic centimeter. The
incubation period was one hour at 37 ° C. The first and third series
in Table I were twice incubated, as this was required by the natureo f the experiments. Then o.1 cubic centimeter of a lO per cent.
suspension of washed human corpuscles and two units of anti-

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Hideyo Noguchi and J. Bronfenbrenner.

95

human amboceptor (rabbit) were added to all tubes, mixed well,and incubated for two hours at 37 ° C. The results are given inTable I.
The foregoing experiments demonstrate several important
points. The first series shows that the addition of various animal
sera, from which the complementary property had disappeared
either by a spontaneous deterioration or by an artificial inactivation,
to the fixing mixture of syphilitic serum and antigen, saturated
completely the latter's fixing capacity so that it was no longer capableof fixing the complement of fresh guinea pig serum subsequently
introduced. Exceptions were encountered with human and goatsera, which behaved inconstantly. While some of the human sera
interfered with the fixation to the same degree as that of mostanimal sera, others showed but little interference. The serum of
the goat was also comparatively less interfering. The most remarkable fact in the series is that the egg-white acted in the same way
as most animal sera.
In the second series we observe somewhat less interference offixation by the sera and egg-white. This is not at all surprising,
because of their simultaneous addition with the complement, underwhich condition the fixing had been directed to both. In the thirdseries there was no hemolysis. It is evident that the subsequent
addition of these substances had no reversing effect upon the complement, which had been fixed before they were introduced.
The deductions which may be drawn from these observations
are that the fixing capacity of the syphilitic serum and antigen canbe saturated not only by active complement-containing sera or inactive sera, but also by an apparently indifferent suspension of proteins (egg-white), so long as their physico-chemicat properties are
the same. It also shows how erroneous it is to estimate thefixing capacity by the estimation of one biological property (complementary) without reference to the ratio of this biological activity to the other components of the serum serving as complement.
For quantitative work with the complement fixation test, the establishment of a standard ratio between the complementary unit andthe volume of the serum, becomes essential. In addition to this,96Interference in Complement Fixation.

the relation of the fixability of the sera of different species must
be considered.

The Complement Fixation by Specific 2Precipitate' (BordetGengou Phenomenon).--ParaUel series of experiments with theforegoing were also made with the precipitate produced by the antimeningococcic horse serum (Flexner and Jobling) and meningococcic extract.
The results obtained were in perfect harmony with those of thesyphilis reaction just described; hence protocols are not called for.
The fixability of these sera and egg-white diminished progressively at temperatures above 560 C., and disappeared at 85 ° C.
Alcohol, which coagulates proteins, removes the interfering property of sera and egg-white.
CONCLUSIONS.

The fixing property of a specific precipitate and of syphilitic
serum in the presence of certain antigenic lipoids, can be removed
by adding certain non-complementary proteins of blood serum or
hen's egg.
This disappearance of the complementary activity in the syphilis
reaction, as well as in the true Bordet-Gengou reaction, is a phenomenon which incidentally accompanies the fixation of certain
serum constituents, some of which possess a complementaryactivity. The presence or absence of the complementary property
in these protein components does not influence fixation. Whether
the disappearance of the complementary activity during the phenomenon of so-called fixation is due to a mechanical precipitationof the molecules through absorption or whether it is due to aphysico-chemical alteration of the active molecules, is unknown. Itis more probable that a chemical interaction takes place in the caseof the syphilis reaction. Certain sera, for example, those derived
from man and goat, show a low fixability.
It is interesting to note that the fixability is gradually diminished when these sera and egg-white are heated to a temperature
above 560 C., and totally disappears at 85 ° C. The coagulation ofproteins with absolute alcohol or by boiling, destroys their interfering property.

Hideyo Noguchi and J. Bronfenbrenner.

97The fact that the fixation is not selectively directed towards complement, has a very important meaning for exact serology. The
one-sided accuracy as to the complementary unity is no longer sufficient for quantitative work. Both the complementary and thevolumetric unity of a serum serving as the source of complement
should be taken into consideration. Besides, the fixability of thesera of various species of animals must also be considered.
F r o m these facts a formula may be derived for deciding" thedegree of suitableness of a serum.

X = K P.
X is the degree of suitableness; K , the species constant for thefixability; P, the complementary activity; and V, the volume ofserum. It will be seen that tile suitableness is proportional to thefixability constant and the complementary unity, and inversely proportional to the volume of serum employed.
As to what species yields the largest' value for X, we refer thereader to our studies published elsewhere. ~(I)Variations in the Complement Activity and Flxabillty of Guinea pigSerum; (2) Comparative Merits of Various Complements and Amboceptors inthe Serum Diagnosis of Syphilis, this number of the Journal o~ Experimental
Medicine.