野口英世著

Journal of Experimental Medicine

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T H E C O M P A R A T I V E M E R I T S OF V A R I O U S C O M P L E M E N T S AND A M B O C E P T O R S IN T H E S E R U M
D I A G N O S I S OF S Y P H I L I S . 1
BY 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 N E R .

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

When using a hemolytic antihuman amboceptor produced in thegoat, one of us (Noguchi)2 found that goat complement could not
be deviated or fixed by the precipitate formed by mixing an extractof Diplococcus intracellularis (Weichselbaum) with antimeningococcic serum (Flexner-Jobling), or by mixing sera of man or different animals with their specific precipitins (rabbit), or egg-whitewith its specific precipitin. Under similar conditions, goat complement can not be deviated in Noguchi's method of the syphilis
reaction. The amb0ceptor from the goat seemed to have astronger affinity for the goat complement and liberated the complement after it had been fixed. Noguchi has also repeatedly shown,in the Bordet-Gengou phenomenon or in the syphilis reaction, thatrabbit complement is less firmly fixed than guinea pig complement.
As he was using a rabbit amboceptor, he could not determine
whether the difficulty in fixing this complement was due to the useof an homologous amboceptor (from the same species) or to theinferior fixing qualities of the complement itself. In order to determine this point, it was necessary to prepare amboceptors fromdifferent species of animals. The preceding problem, together withothers concerning the comparative merits of the complements ofvarious species of animats~in the complement fixation phenomenon,
the Bordet-Gengou, and the Wassermann reactions, is consideredin this communication. The questions just outlined include thedeterminations of the complementary activity of the sera of various
1Received for publication, September 21, 19IO

*Proc. Soc. Exper. Biol. and Med., 191o, vii, 55-78

Hideyo Noguchi and J. Bronfenbrenner.

79

species of animals with homologous and heterologous amboceptors.
They also bring out the relative capacities of different species ofanimals for producing hemolytic amboceptor for human erythrocytes. Apart from the scientific interest, we intended also to search
for a suitable gpecies of the larger domestic animals whose complement or amboceptor would yield results as reliable as those obtained with guinea pig complement and rabbit amboceptor. If thisshould be accomplished, an economy would result.
PREPARATION OF ANTII-IUIVIAN AI~BOCEPTOR.

In our experiments the washed human erythrocytes suspended ino. 9 per cent. salt solution, corresponding with the original volumeof the blood, was used for immunizing the animals. The varietiesof animals used and the quantities of erythrocytes injected are
given below :Dog, subcutan,eous injection,
20, 30, 30, 30 c.c.
Cat, intraperitoneai injection,
5, IO, 15, 2o c.c.
Rabbit, intraperitoneal injection
5, Io, 15, 2oc.c.
Guinea pig, intraperitoneal injection,, 2, 3, 5, 7 c c .
Rat, infraperitonealinjection,
I, 2, 3, 3c.c.
Chicken, in~raperitoneal injection,
3, 4, 5, 5 c c .
Goat, a subcutaneous injection.

With the exception of the goat, more than two animals ofeach of the species were employed, and several of each of thesmaller animals. Injections were made four to five days apart,and the serum was collected on the ninth day after the last injection. These amboceptoric sera were inactivated at 56o C. forthirty minutes. After inactivation, the strength of amboceptor ofeach of the above animals was determined by titration with homologous and heterologous complement. The complements employed
were from dog, cat, rabbit, guinea pig, rat, chicken, goat', pig, sheep,
and ox. Owing to the small quantity of blood that could be obtained from the immunized rats, the amboceptor from this animal
could not be titrated to the extent of the others.
The amount of complement of each species used was uniformly
0.0 5 cubic centimeter for each tube. The erythrocytic suspension
This goat was g i v e n about twenty subcutaneous injections.

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82

The Serum Diagnosis of Syphilis.

was employed in a dose of o.I cubic centimeter of a IO per cent.
suspension of washed human corpuscles. The total volume in eachtube was made uniformly one cubic centimeter.
The above experiments revealed that a striking relation exists between the various complements and a given kind of amboceptor produced in different species of animals.
This is brought out in Table II.
With the amboceptors produced in guinea pig, cat, and chicken,
the strongest action was obtained with their homologous complements. On the other hand, the amboceptors from the dog and rabbit
acted more strongly with certain heterologous complements thanwith homologous complements. It was not the same heterologous
complements, however, which showed this peculiarity. The strongest action with rabbit amboceptor was obtained with the complements of guinea pig, goat, cat, and dog. The complementary actionof rabbit serum with dog amboceptor was, however, very muchstronger than that of the dog complement with the amboceptor obtained from the dog. This dog amboceptor was thirty times more
active in the presence of goat complement than in the presence ofdog complement. Guinea pig complement, which has hitherto been
considered universally suitable, is seen to be inferior to the complements of goat, rabbit, and pig when used with dog amboceptor, andit was without any complementary action for chicken amboceptor.
The complements of ox and sheep, which were found to be quite
suitable for goat amboceptor, are seen to be less suitable for theamboceptors derived from rabbit, guinea pig, dog, cat, and chicken.
It is also remarkable that the sera of all mammals, except the pig,
used in our experiments are devoid of any complementary action
for chicken amboceptor, while chicken serum contains certain
amounts of complement suitable for the amboceptors derived fromthose mammals.
Thus we have learned that the titers of an amboceptor may varyconsiderably according to the varieties of sera employed as complement. That the variations revealed in the titration of a given
amboceptor are due to the variations in the complement content of
the serum used as complement, can be further demonstrated by determining the limit of activity of the sera (serving as complement)

Hideyo Noguchi and J. Broufenbrenner.

83

in the presence of a constant amount of amboceptor. In the following table is shown the comparative activity of different sera-ascomplement. Rabbit amboceptor (in excess) was employed in thisexperiment.
TABLE In.
Titration of sere. as complement with antlhuman rabbit amboceptor.

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Table I I I shows that the titration of the complementary activityof the sera of guinea pig, cat, arid dog is about twice as strong asthat of the rabbit for rabbit amboceptor. In this respect sheep
serum was about sixteen times weaker, and chicken serum was t o oweak to produce complete hemolysis.
We have also studied the complementary activity of various sera
for the amboceptors derived from heterologous as well as fromhomologous species. Our results are given in TaMe IV.
This series of experiments demonstrates three interesting facts:( I ) the preference of certain amboceptors for their homologous
complements (chicken, guinea pig, goat); (2) the inferiority ofcertain complements for homologous amboceptors (rat) ; and (3)the increase of hemolytic activity of complements in general by theuse of a larger amount of the amboceptor. The inferiority ofhomologous complements to certain heterologous complements has
already been described with the sera derived from rabbits and dogs
(see Table II). That cat amboceptor is more powerful with its
homologous complement is shown in the same tame. Thus, we findthat the amboceptors derived from chicken, guinea pig, goat, andcat are stronger in the presence o~ their homologous complements,

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Hideyo Noguchi and J. Bronfenbrenner.

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while those from rabbit, dog, and rat are stronger in the presence of
certain suitable heterologous complements.
After studying the complex inter-relation of various complements and amboceptors, we took up the further study of a few complements. The sera of pig, sheep, and ox were studied especiallybecause we hoped to be able to utilize them as complement, for they
may be obtained in large quantity from any abattoir. We soon discovered, however, that they were quite unsuitable for the syphilis
reaction. In the first place, ox serum often contains a variable and sometimes considerable amount of natural amboceptor
for human erythrocytes, and for this reason we did not pursue thestudy of this serum.
The sera of pig and sheep are usually devoid of the natural antihuman amboceptor. They were examined for their complementary
activity for rabbit amboceptor. The results are shown in Tables
V and VI.
The striking feature of the sera of these two animals is the rapid
disappearance of their complementary activity. It is more marked
with pig serum. Both varieties of serum became so weak after ~forty-eight hours (o ° C.) that they no longer produced complete.
hemolysis in a quantity as large as o.I cubic centimeter. Guinea pig
serum, under the same conditions, showed practically no diminution
during the same period of preservation. The rapid deterioration ofboth pig and sheep serum renders them unreliable because of theconstantly decreasing activity af complement. The rate of deterioration is also quite irregular with different samples of the same
variety of serum. Thus, in point of reliability and activity, they are
far inferior "to the serum of the guinea pig and are not recommended
for use in the syphilis reaction.
CONCER2qI•G

THE

U S E O F V A R I O U S SIRRA AS C O M P L E ~ E ~ q T I N TI-I~.:
SERUM

REACTIOIVOFSYPHILIS.

In deciding whether or not the serum of a given animal is suitable
as complement in the complement deviation test, we have to consider two essential properties of the serum. The first is, of course,.
its complementary activity, and the second is its susceptibility to,
fixation. "We have already studied in detail the comparative corn-

86

The Serum Diagnosis of Syphilis.
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The Serum Diagnosis of Syphilis.

plementary activities of the sera of various species of animals. W epropose to report here, therefore, the results which we have obtained with regard to their comparative fixability.
As mentioned at the beginning of this article, Noguchi long ago
showed that the complement o f goat serum remains undeviatedby specific precipitates or by the syphilis reaction. Thisphenomenon was decidedly more pronounced when the goat furnished both complement and amboceptor. He has also demonstrated that rabbi¢ serum is less sensitive to fixation than guinea pig
serum. Hence he suspected that the amboceptor might have sucha strong affinity toward its homologous complement as to reverse
partially the process of fixation. The question of whether or notthere is such a relation between the source of amboceptor and thefixabitity of complements was left unsettled. This was one of the
problems which we set out to solve, and our results will now begiven.
The fixation test was carried out with Noguchi's system in theusual way. The positive serum was from a case of untreated secondary syphilis, and, in the quantity of 0.002 cubic centimeter, wascapable of fixing completely o.04 cubic centimeter of guinea pig
serum. In the following experiments, 0.02 cubic centimeter of thesyphilitic serum was used for each tube.
TABLE

VII.
COMPLEMENT.

Guinea pig o.o4
(2 units)'.

l~abblt I~,To.x O.O8 l~abblt No. 2(2 units),
o.o$ (2 units).

Goat No. z

0.04 (2 units).

Goat No. 2

0.04(2 umts).

Guinea pig No hemolysis Partial hemolysis Partial he- Complete he- Complete heamboceptor(no hemolysis for molysis (nomolysismolysis(2 units)
some time),
hemolysis for (much delay), (much delay),some time).
RabbitNo hemolysis Partial hemolysis Partial he- Complete he- Complete hemolysis
molysisambooeptor(no hemolysis for molysis (nosome time).
hemolysis for (much delay). (much delay),(2 units).
some time).
Goat amboceptor(2 units).

Slight hemol- M u c h hemolysis M u c h hemol- Complete he- Complete heysis (no he- ( n o hemolysis for ysis (no hemolysis
molysismolysis forsome time).
molysis for(slight(shghtsome hours).
some time),delay).
delay).

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No go

Hideyo Noguehi and J. Bronfenbre~er.

89

The experiments given above show that the complement contained in goat serum is totally unfixable. There was some delay incommencing hemolysis, but it was finally completed within a few
hours. The complement contained in rabbit serum showed thepeculiarity that hemolysis commenced after six hours or so, and after
about twenty-four hours a moderate amount of hemolysis was observed. This is most striking when compared with the complete andlasting inhibition of hemolysis in the case of guinea pig complement.
The use of goat amboceptor, however, has caused more or less hemolysis even with guinea pig complement. Experiments also show thatwhen the complements are fixable the homologous amboceptor andcomplements do not influence the fixation phenomenon. It is difficult to explain why the fixation Pf guinea pig complement is imperfect when tested by goat amboceptor. It may be due to the presence in guinea pig serum of a non-fixable complement, which, while
quite active with the goat amboceptor, is inactive with the amboceptors from rabbit and guinea pig. Now, with regard to the state ofnon-fixation of the complement of goat serum, there are two possibilities. The first is its insensi,tiveness to fixation. The delay incommencing hemolysis may be accounted for by assuming that thequantity of complement still free in the mixture was considerably
reduced by a partial fixation. The second is the gradual liberationof complement already fixed through the introduction of the goat
amboceptor. Apart from these hypothetical considerations, theabove experiments showed that rabbit and goat complement andgoat amboceptor are unsuitable in deviation tests.
We have determined the fixability of the complements containedin the sera of pigs and sheep. Pig complement was fixable, whilethat of sheep was somewhat less so. But, as has already been
pointed out, their complementary activity is so feeble that, in order
to employ two units, their absolute quantities may reach o.i cubic
centimeter or even exceed this. If the fixation were selectively
directed to the complement, an accurate result could always be
secured by employing any quantity in which two activity-units
existed. But as we shall describe elsewhere, the fixation is notselectively directed to the complement. The presence of non-complementary protein constituents may, therefore, interfere with the

90

The Serum Diagnosis of Syphilis.

fixation of complement. From this it will be easy to understand
why we must avoid weak sera, for in these the quantity which contains a single unit of complement is too large.
CONCLUSIONS.

I. The maximum activity of an antihuman hemolytic amboceptor may be obtained by employing the homologous or heterologous
complement, according to the variable relations existing between
the species furnishing the amboceptor and the one supplying thecomplement. Thus, some amboceptors are best reactivated by thecomplement of the same species, while others may act most strongly
when reactivated with the complements of certain suitable heterologous species.
2. From the above it is clear that the complementary activity ofa given Serum may be very variable according to the varieties ofamboceptors employed. In expressing the complementary activityof a serum, the species of the host of the amboceptor must always
be stated. Thus, one serum may have many different complementary titers according to the amboceptors used. A similar variation in the titers of the amboceptors occurs when a variety of complements are employed.
3- Certain species of animals (pig and sheep) yield sera which
are comparatively poor in reactivating most varieties of antihumanamboceptors. The complements of these species deteriorate rapidly.
4- The serum of chicken contained but little complement for theamboceptors derived from the mammalia, while the amboceptor
from the chicken Was only poorly, or not at all, reactivable by thecomplements contained in the mammalian sera. The serum of pig
was the only variety which reactivated this amboceptor in a fairdegree.
5- For the fixation tests guinea pig complement is the mostfavorable. This complement is also the most active and durable ofthose which have been studied, The complements of pig and sheep
are quite fixable, but their weakness and rapid deterioration renderthem unsuitable for fixation purposes. Rabbit complement is quite
active but is not easily fixable. Goat complement is, as alreadyHideyo Noguchi and J. Bronfenbrenner.

91

stated, difficult to fix, in spite of its strong complementary activity.
The other complements are unsuitable because of their feeble complementary activity.
6. For fixation tests the antihuman amboceptors produced in the
rabbit and guinea pig are suitable. They are, moreover, very active
and do not cause the phenomenon of non-fixation. The amboceptors from other animals are unsuitable, as we cannot find a complement which strongly reactivates them. The amboceptor from thegoat is unsuitable because of the danger of masking the fixation
phenomenon by subsequent hemolysis.
7. In summing up, we arrive at the conclusion that the rabbit is
the best animal for producing antihuman amboceptor, and theguinea pig for supplying complement. The guinea pig produces agood amboceptor, but its small size renders it second in choice.
So far, no other animals have been found useful for the fixation
experiments.