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A MODIFICATION OF ROEMER'S INTRACUTANEOUS
METHOD FOR THE DETERMINATION OF SMALL
AMOUNTS OF DIPHTHERIA ANTITOXIN
IN BLOOD SERA *
Abraham Zingher
From the Research Laboratory of the Department of Health, New York City
A convenient, practical, and economical test for the determination
of the diphtheria-antitoxin content of blood sera is important for a
number of reasons, among which may be mentioned the desirability of
estimating quantitatively the efficiency of active immunization with
toxin-antitoxin, the development of an active immunity after an attack
of diphtheria, the antitoxin content of the blood at varying intervals
after a single therapeutic or prophylactic injection of diphtheria anti-
toxin, the effect of one injection of antitoxin on the duration of
immunity conferred by a second injection, etc. Such determination of
the antitoxin content would help to give us a solid scientific foundation
in many experimental and clinical problems connected with the sub-
ject of diphtheria.
The older subcutaneous test of Ehrlich for determining the anti-
toxin content by the death of the guinea-pig has been universally
accepted as a standard, but it cannot be used with any degree of
accuracy when less than 1/20 of a unit of antitoxin to the cubic centi-
meter of serum is to be determined. The subcutaneous test requires
a large amount of serum for each test, especially when the test is made
for a fraction of a unit of antitoxin. This amount is not always obtain-
able in the case of human beings, especially when we are dealing with
small children. In addition, the subcutaneous test is expensive in that
it requires the use of one guinea-pig for each test. A method, there-
fore, that would enable us accurately to determine as little as 1/200
unit of antitoxin, require but a small amount of serum, and be saving
of animals, would be of considerable advantage. When a larger series
of sera is to be tested for small amounts of antitoxin, the intradermal
method is practically the only feasible one.
Recent work in active immunization against diphtheria 1 has shown
successful results in producing an antitoxic immunity in 90-95% of
* Received for publication May 7, 1916.
1 Park and Zingher: Jour. Am. Med. Assn., 1915, 65, p. 2216. Am. Jour. Pub. Health.
1916, 6, p. 431.
558 Abraham Zingher
susceptible individuals. This was shown by negative Schick reactions
in individuals who had previously given positive reactions. The nega-
tive Schick reaction, however, indicates only that the individual has
become immune by the development of 1/30 of a unit (according to
our work 1/100 unit) or more of antitoxin to the cubic centimeter;
it does not show the exact amount of antitoxin that has been produced.
Altho clinically it is possible to demonstrate the continuation of the
immunity by repeating the Schick test at definite intervals, yet for
estimating the duration of immunity it is important, according to
v. Behring, to establish quantitatively the amount of antitoxin produced
in the actively immunized persons.
In 1909 Romer and Sames 2 published a method for testing small
amounts of antitoxin in blood sera. This test was based on a previous
observation made by Romer 3 that 1/500 M. L. D. of a toxin was still
capable of producing a distinct local necrosis when injected intrader-
mally into the guinea-pig.
Romer and Sames determined the necrosing dose of a toxin in the
guinea-pig — for example, the amount of toxin added to one unit, or
any fraction of a unit of antitoxin, which is still capable of producing
a slight local necrosis. This was called the Ln dose of the toxin. They
found that 1/500 M. L. D. of the toxin was sufficiently neutralized by
1/40,000 unit of antitoxin to just produce a slight local effect in the
animal. This amount of antitoxin in 1/20 c.c. of undiluted serum (the
quantity used in the test with an equal volume of toxin solution)
represents 1/2000 unit of antitoxin to the cubic centimeter.
The method suggested by Romer and Sames and later applied by
Romer and Somogyi 4 to the testing of concentrated antitoxic horse
sera, has worked satisfactorily in their own hands, but has not given
reliable results in the hands of others who have tried it repeatedly.
One of the difficulties lies in their attempt to make too fine a distinction
between the various lesions of closely lying tests. They claim to be
able to distinguish a 5% difference in antitoxin content between the
individual tests. After repeated trials this has seemed to me difficult
and unnecessary, especially when we are dealing with an antitoxin
content between 1/200 unit and 10 units to the cubic centimeter of
serum. A second difficulty and an equally unnecessary point is the
long time, 24 hours, that the toxin and serum are required to stand
before injection into the animal. Thirty minutes has proved to be a
sufficient time to allow for the full union between toxin and antitoxin,
2 Ztschr. f. Immunitatsf., 1909, 3, p. 49.
3 Ibid., p. 208.
4 Ibid., p. 433.
Modification of Intracutaneous Test of Antitoxin 559
even in the dilutions used in the test. A third difficulty lies in the
necessity of establishing a new toxin test dose, the Ln. These factors
have undoubtedly discouraged the use of a test which is inherently
valuable.
The modification which I have found simple and workable in the
testing of many hundreds of human and animal sera, has the following
advantages :
( 1 ) The L -f- of the toxin is taken as the test dose and so diluted
that each cubic centimeter represents 1/100 L + dose.
(2) Two test points are established with each serum, the test at
which no lesion develops, and the one for twice the amount of antitoxin
at which a distinct lesion appears. Within these two limits lies the
antitoxin content of the serum. The first test indicates a balanced
combination or a slight excess of antitoxin, while the second shows
the presence of a trace of free toxin. The latter test which produces
a slight local necrosis represents the antitoxin content of the serum.
When the necrosis is more marked, an intermediate test is made.
Example: Slight necrosis at 1 unit, no necrosis at 1/2 unit. Antitoxin
content 1 unit. If necrosis were marked, the test would be repeated
for 3/4 unit.
(3) It saves animals in that 4 tests can be made on each guinea-
pig. It also saves time, for the tests can be interpreted in from 48 to
72 hours.
(4) As little as 1/200 unit of antitoxin can be determined in a
serum with a fair degree of accuracy. If a distinct lesion appears at
this test point, the serum is considered as having no antitoxin.
Details of the Test
(1) A standard well-ripened toxin is used, so diluted with normal salt solu-
tion that 1 c.c. represents 1/100 L + dose. A fresh dilution is prepared for
each set of tests. If, for example, the L + is 0.5 c.c, then that amount is
diluted with 99.5 c.c. of sterile salt solution. The quantity of toxin added in
the test varies with the antitoxin content for which the test is made (see
Table 1).
(2) The serum to be tested is used either undiluted or diluted 1 : 10 (0.2 c.c.
serum + 1.8 c.c. salt solution), 1:100 (0.2 c.c. of serum dilution 1 : 10 +
1.8 c.c. salt solution), etc. The amount of serum used in the test is always
0.2 c.c, except when the test is made for 1/200 unit of antitoxin; then 0.4 c.c.
of undiluted serum is added to 0.2 c.c. of toxin solution.
(3) Normal salt solution is added to balance the mixture. The amount is
the same as that of the toxin solution minus 0.2 ex., which represents the serum.
(4) After the addition of toxin, serum, and salt solution, the mixture is
allowed to stand for 30 minutes at room temperature before it is injected
into the guinea-pig. This time is utilized in preparing and tagging the
guinea-pigs.
560
Abraham Zingher
(5) The guinea-pigs (300-350 gm.) are prepared by removing the hair from
the abdomen. This is best accomplished by simply pulling it out, a central
line of hair being left to divide the abdominal surface into halves. The pull-
ing of the hair is easily and rapidly carried out, and is probably less painful
than the action of a depilatory like calcium hydrosulfid or shaving of the hair.
The first occasionally leads to local eczema, the second to abrasions that inter-
fere with the reading of the test.
(6) The animal holder is simply an elevated board with 4 openings for
loops of cord with which to fasten the limbs of the animal. This simple
device is essential in accurate work and facilitates the exact placing of the
injections.
(7) A 1-c.c. "Record" or tuberculin syringe and a fine steel or platinum-
iridium needle are necessary.
(8) Four injections are made into each guinea-pig, the abdomen being for
this purpose divided into right upper (R. U.), right lower (R. L.), left upper
(L. IL), and left lower (L. L.) quadrants. The injections are made as far
apart as possible to avoid a fusion of the lesions. Two-tenths cubic centi-
meter of each test mixture is injected intradermally. Care with each injection
is essential if uniform results are to be obtained. A good guide in the insertion
of the needle into the proper layer of the skin is to be able to see its oval
opening through the superficial layers of the epidermis. If the fluid has been
injected properly, a tense local sharply circumscribed swelling appears, which
shows the prominent openings of the hair follicles.
(9) The places of injection on the guinea-pigs are examined at the end of
24, 48, 72, and 96 hours, and a careful record is kept of each test (Table 3).
The following changes in the local appearance of the skin at the sites of
injection are noted:
(a) Redness, whether marked (R) or slight (r).
(b) Induration, whether marked (Id), moderate (mod), or slight (si).
(c) Necrosis, marked (N) or slight (n), or only superficial scaling (seal).
The induration is most marked at the end of 48 or 72 hours; the necrosis
appears at the end of 72 hours.
The following tables show (1) the amounts of toxin, serum, and
salt solution used in the test; (2) the method followed in the examina-
tion of several sera; (3) a sample page of the daily record of the tests
on the guinea-pig; and (4) an experiment on 4 horses showing the
development of antitoxin after injections of small amounts of diph-
theria toxin-antitoxin. table i
Intradermal Estimation of Diphtheria Antitoxin
Test Toxin
1
le.c. = — L+
100
Normal
Salt
Solution
Serum
=
Undiluted
1:10
1:100
1:1000
1:10,000
0.2 + 0.0 + 0.2
0.01 unit
0.1 unit
lunlt
10 units
100 units
0.4 + 0.2 + 0.2
0.02 unit
0.2 unit
2 units
20 units
200 units
1.0 + 0.8 + 0.2
0.05 unit
0.5 unit
5 units
50 units
500 units
2.0 + 1.8 + 0.2
0.10 unit
1.0 unit
10 units
100 units
1000 units
Every 0.2 c.c. ot toxin solution added is
0.01 unit
0.1 unit
lunit
10 units
100 units
Modification of Intracutaneous Test of Antitoxin 561
table 2
Estimation of Diphtheria Antitoxin by the Intradermal Method
OTlKBA-m 1
WIHIA-PIO Z
0UIXIA-PIS 3
GUINEA-PIO H
UU LJU
CZU CO
1 ? I | p |
nrj i_b i
» U W 10
Eg
Iwl (HTil
El E3
lLs_l LxJ
*o mt
^O OJ
1 • O l
o o
2 O ^100
* • o*
2 # Q2
A -1/10+ C-l/10-
• -1/10 + B*l/10+
A =.1/2+ B* 1/100-
0=1/2 B = 1/2+
A=l B--2 +
ODIKU-PIt 5
ssniu-PM <
CO L_LJ
io O O io °
WIMA-PIO 7
SUINU-P1S S
LJJ LaJ
Standard
antltoxii
Standard
antitoxic
5 o*
E3
03
"• # l
l O *.i
l m # 2
* # # l
B -- 10 A = 1
I si* 7«{l/100 +
IVlo-
1 c.c.»l unit
1 c,c>l unit
Rectangles indicate blood sera and their dilutions. Circles indicate results of tests, the
white ones meaning no lesion, the black ones local necrosis, and the shaded ones slight local
inflammatory lesions.
TABLE 3
Sample Page of Daily Record of Tests
Guinea-
pig
Place
of
Injection
Amount ol
Antitoxin
Tested
Daily Observations
Result
ol
Tests
Feb. 13
Feb. 14
Feb. 15
Feb. 16
A
1
EC
1/10 unit
OK
OK
OK
OK
1/10 +
B
1
BL
1/10 unit
SI
r
Mod
R
Idn
R
IdN
R
1/10 -
O
1
LU
1/10 unit
OK
OK
OK
OK
1/10 +
C
1
LI
1/5 unit
SI
r
Mod
R
Mod n
R
Mod n
R
1/5
R U, R L, LU, L L refer to the right upper, right lower, left upper, and left lower
quadrants, respectively, of the guinea-pig abdomen.
OK = no reaction; SI = slight induration; Mod =r moderate induration; Id = marked
induration. N = marked necrosis; n = slight necrosis. R = marked redness: r = slieht
redness. '
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Modification of Intracutaneous Test of Antitoxin 563
Guinea-pig 1 (Table 2) shows the preliminary results of tests on
4 different sera for 1/10 unit of antitoxin. Sera A, C, and D com-
pletely neutralized the toxin solution and no lesions appeared. Serum
B gave a necrosis. Result: A, C, and D had more than 1/10 unit, B
less than 1/10 unit of antitoxin to the cubic centimeter.
Guinea-pigs 2 and 3 represent the 2nd set of tests on the 4 sera.
Serum A showed no lesion with the 1/2-unit test, therefore more than
1/2 unit was present. Serum B showed less than 1/100 unit, a necrosis
appearing with this test. The serum can be further tested for 1/200
unit and if a definite lesion appears the serum is considered as having
no antitoxin. Serum C showed no lesion with 1/5 unit, but a slight
lesion with 1/2 unit. Antitoxin content therefore was 1/2 unit to the
cubic centimeter. If a marked lesion had developed, an intermediate
test for 1/3 unit could be made. Serum D had more than 1/2 unit.
Guinea-pig 4 represents the 3rd set of tests for Sera A and D.
Serum A showed a lesion with tests for 1 and 2 units. The antitoxin
content was therefore 1 unit, since the test in Guinea-pig 2 with
Serum A for 1/2 unit showed no lesion. Serum D showed more
than 2 units.
Guinea-pig 5 represents the 4th set of tests for Serum D. A lesion
appeared with the test for 10 units and no lesion with the one for
5 units. The antitoxin content therefore equaled 10 units. Serum A
was retested for 1/2 and 1 unit to show the nonnecrosing and necros-
ing test points on the same animal.
By using 5 animals we were able to make 20 different tests, and
arrive accurately at the antitoxin content of 4 different sera.
Guinea-pig 6 shows 2 sera, E and F, on which the tests were made
for widely separated antitoxin contents. This can be done when we
wish to arrive rapidly at rough limits of the amount of antitoxin in
a serum. Serum E showed more than 1 unit, Serum F more than
1/100, but less than 1/10 unit. Serum E would have to be tested
further for 10, 100 units, etc., while Serum F should be tested for
1/20 and 1/50 unit, if we wish to establish the accurate amount of
antitoxin.
Guinea-pig 7 shows tests made by this method on the standard
antitoxin furnished from the Hygienic Laboratory in Washington.
Each cubic centimeter represents exactly one unit of antitoxin. With
the intradermal test the antitoxin content was also definitely estab-
lished as 1 unit to the cubic centimeter, no lesion appeared with the test
for 1/2 unit, but a distinct lesion with slight necrosis with the test for
564 Abraham Zingher
1 unit. The tests for iy 2 and for 2 units showed more marked
necrosing lesions.
Guinea-pig 8 shows the same standard antitoxin tested for 0.7, 0.8,
0.9, and 1 unit. Slight inflammatory lesions were noticed even with
the test for 0.7 unit. It is therefore best not to attempt to make too
fine a differentiation between the slight lesions of closely lying tests,
but to endeavor to obtain the necrosing and nonnecrosing points of a
serum, the first representing twice the amount of antitoxin tested for
in the second. When we are dealing with concentrated antitoxic sera
(100-1000 units to the cubic centimeter) the intradermal test can give
us only a preliminary idea of the antitoxin content ; the more accurate
estimation is subsequently made by the subcutaneous test.
CONCLUSIONS
An intradermal test is presented for estimating in an economical
way small amounts of antitoxin.
The method is easily carried out, tho it requires for accuracy a
uniform technic in making the dilutions of toxin and serums, the allow-
ance of a uniform time period for the two to combine, uniformity in
the method of injection into the guinea-pig, daily observations of the
animals, and uniform interpretation of the tests.
Problems in experimental work in diphtheria and the accurate sero-
logic control of actively immunized individuals can be carried out
by this test.
