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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.