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A STUDY OF COMPLEMENTS USED IN THE 
WASSERMANN REACTION 

E. H. RUEDIGER 

From the Pathological' Laboratory of the Bismarck Hospital, Bismarck, N. D. 

The serums commonly used as complement in the Wassermann 
reaction are those of guinea-pig and of man. That the complement 
content of serums varies has repeatedly been shown and Browning 
and McKenzie 1 found that complements vary in fixability. 

Complements usually deteriorate rapidly and several workers 
sought by artificial means to prevent such deterioration. Noguchi 
dried it on filter paper, others kept it in a frozen state and more 
recently Ramy 2 proposed sodium acetate as a preservative for 
complement. 

The use of human complement has been advocated by Hecht, 3 
Tschernogubow, 4 Fleming, 5 Emery, c von Dungern, 7 Gurd, 8 Noguchi 9 
and others. 

In this report the following points are considered : 

1. Fixability and complement content of fresh guinea-pig serum. 

2. Fixability and complement content of fresh human serum. 

3. Noguchi's recent method compared with our regular method. 

4. The effect of normal human serum on syphilitic human serum. 

5. The effect of normal guinea-pig serum on syphilitic human 
serum. 

6. The rate at which syphilitic serum is altered by normal serum. 

7. Deterioration of guinea-pig complement in the refrigerator. 

8. Sodium acetate as a preservative for complement. 

9. Glycerol as a preservative for complement. 

Received for publication Sept. 20, 1918. 

1 Ztschr. f. Immunitatsforsch. u. exper. Therap. 1909, 2, p. 459. 

2 Jour. Am. Med. Assn., 1917, 69, p. 973. 

3 Wiener klin. Wchnschr., 1909, 22, p. 338. 

* Deutsch. med. Wchnschr., 1909, 35, p. 668. 

Lancet, 1909, 1, p. 1512. 

6 Lancet, London, 1910, 179, p. 732. 

' Miinch. med. Wchnschr., 1910, 57, p. 507. 

8 Jour. Infect. Dis., 1911, 8, p. 427. 

9 Jour Am. Med. Assn., 1918, 70, p. 1157. 



Complements in Wassermann Reaction 



121 



Method 

In our regular method the serum to be tested is heated to about 55.5 C. 
for 30 minutes and is mixed with an equal volume of sterilized glycerol. Six 
test tubes are used and each receives 0.2 c c of serum-glycerol mixture. Com- 
plement is used in quantities of 0.2 c c of a 1:5, 1 : 10 and 1 : 20 dilution. 
Of antigen, alcoholic extract of human heart muscle, the largest dose that is 
no longer anticomplementary, is used and is diluted so that the test dose 
is contained in 0.2 c c. Human corpuscles are well washed, the washed blood 
corpuscles are made up into a 2.5% suspension in salt solution (0.9%), and 
the test dose is contained in 0.2 c c of the suspension. Hemolytic amboceptor 
is prepared in rabbits and is used in doses of 1 unit per test tube. The 
term unit is applied to the smallest quantity which with 0.2 c c of 1 : 10 
dilution of complement in the presence of 0.2 c c of serum-glycerol mixture 
completely dissolves the test dose of blood corpuscles in one hour. The total 
quantity in each test tube is 1 c c, first incubation is in the refrigerator for 
5 hours and second incubation in the incubator for one hour. After the tubes 
have stood at room temperature for from 1-2 hours the results are read 
and recorded. 

Test 1 

Ten guinea-pig serums, Serums 1-10, inclusive, were tested on 12 syphilitic 
human serums. All complement serums were used singly and in mixtures 
of 5 serums each. The quantity of amboceptor was kept uniform throughout. 



TABLE 1 

Comparison of Ten Guinea-Pig Complements 













Readings 










Number 
of 


Sex 
ol 


Ambo- 
ceptor 














Xumber 


Antigen 


Control 




of 


Co i ple- 


Guinea- 


per Tube 


' 


rubes 


Tubes 


Results 


Serum 


ment 


Pig 


Unit 














1 


2 


3 


V 


9/ 


3' 




1 


1 


M 


lg 


+ 








+ 


+ 


-t- 


Strongly positive, 5+. 




2 


F 


lg 


+ 


tr 





+ 


+ 


-t- 


Strongly positive, 4+. 




3 


M 


lg 


+ 








+ 


+ 


tr 


Strongly positive, 4+. 




4 


F 


lg 


+ 








+ 


+ 


-4- 


Strongly positive, 5+. 




5 


M 


lg 


+ 








+ 


+ 


-t- 


Strongly positive, 5+. 




1- 5 


M and F 


lg 


+ 








+ 


+ 


■+■ 


Strongly positive, 5+. 




6 


F 


lg 


+ 


tr 





+ 


+ 


■+• 


Strongly positive, 4+. 




7 


M 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 4+. 




8 


F 


lg 


+ 


tr 





+ 


+ 


± 


Strongly positive, 4+. 




9 


M 


lg 


+ 


-t- 





+ 


+ 


+ 


Strongly positive, 3+. 




10 


F 


lg 


+ 


tr 





+ 


+ 


-+■ 


Strongly positive, 4+. 




6-10 


M and F 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 4+. 


' 


1 


M 


lg 


+ 


-f- 





+ 


+ 


■+■ 


Strongly positive, 3+. 




2 


F 


lg 


+ 


-t- 





+ 


+ 


■+■ 


Strongly positive, 3+. 




3 


M 


lg 


+ 


-*- 





+ 


+ 





Weakly positive, 1+. 




4 


F 


lg 


+ 


+ 





+ 


+ 


-t- 


Moderately positive, 2+. 




5 


M 


lg 


+ 


+ 





+ 


+ 


-+■ 


Moderately positive, 2+. 




1- 5 


M and F 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




6 


F 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




7 


M 


lg 


+ 


+ 





+ 


+ 


-+• 


Moderately positive, 2+. 




8 


F 


lg 


+ 


+ 





+ 


+ 


-4- 


Moderately positive, 2+. 




9 


M 


lg 


+ 


■+■ 





+ 


+ 


-+- 


Strongly positive, 3+ . 




10 


F 


lg 


+ 


■+• 





+ 


+ 


-+- 


Strongly positive, 3+. 




6-10 


M and F 


lg 


+ 


+ 





+ 


+ 


-+- 


Moderately positive, 2+. 



* Explanation: In all tables + = complete hemolysis; 
100%; tr = hemolysis less than 50%; = no hemolysis. 



= hemolysis between 50% and 



122 



E. H. RUEDIGER 



TABLE 1 — Continued 
Comparison of Ten Guinea-Pig Complements 













Readings* 










Number 
of 


Sex 
of 


Ambo- 
ceptor 














Number 


Antigen 


Control 






of 


Comple- 


Guinea- 


per Tube 


Tubes 


Tubes 




Results 




Serum 


ment 


Pig 


Unit 














1 


2 


3 


V 


2' 


3' 






3 


1 


M 


lg 


+ 








+ 


+ 


-f- 


Strongly positive, 


54-. 




2 


F 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


54-. 




3 


M 


lg 


4 


tr 





+ 


+ 





Moderately positive, 2+. 




4 


P 


lg 


4- 


4- 





+ 


+ 


+ 


Strongly positive, 


34-. 




5 


M 


lg 


+ 


tr 





+■ 


+ 


+ 


Strongly positive, 


44-. 




1- 5 


M and P 


lg 


+ 


tr 





+ 


+ 


4- 


Strongly positive, 


44-. 




6 


F 


lg 


+ 


-+- 





T 


+ 


+ 


Strongly positive, 


34-. 




7 


M 


lg 


+ 


-+- 





+ 


+ 


+ 


Strongly positive, 


34-. 




8 


F 


lg 


+ 


4- 





+ 


+ 


+ 


Strongly positive, 


34-. 




9 


M 


lg 


+ 


-+- 





+ 


+ 


+ 


Strongly positive, 


34-. 




10 


P 


lg 


+ 


tr 





+ 


+ 


~h 


Strongly positive, 


44-. 




6-10 


M and P 


lg 


+ 


± 





+ 


+ 


± 


Strongly positive, 


34-. 


4 


1 


M 


lg 


-1- 


tr 





+ 


+ 


-+- 


Strongly positive, 


44-. 




2 


F 


lg 


+ 


4- 





+ 


+ 


+ 


Strongly positive, 


34-. 




3 


M 


lg 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




4 


P 


lg 


+ 


tr 





+ 


+ 


■+■ 


Strongly positive, 


44-. 




5 


M 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


44-. 




1- 5 


M and F 


lg 


+ 


tr 





+ 


+ 


-t- 


Strongly positive, 


4-f. 




6 


F 


lg 


+ 








+ 


+ 


■+• 


Strongly positive, 


5 + . 




7 


M 


lg 


+ 








+ 


+ 


+ 


Strongly positive, 


54-. 




8 


F 


lg 


+ 








+ 


+ 


-t- 


Strongly positive, 


5+-. 




9 


M 


lg 


+ 








+ 


+ 


± 


Strongly positive, 


54-. 




10 


F 


lg 


4- 








+ 


+ 


± 


Strongly positive, 


5-I-. 




6-10 


M andF 


lg 


+ 








+ 


+ 


+ 


Strongly positive, 


5 + . 


5 


1 


M 


lg 


+ 


4- 





+ 


+ 


+ 


Strongly positive, 


34-. 




2 


F 


lg 


4- 


+ 


tr 


+ 


+ 


+ 


Moderately positive, 24-. 




3 


M 


lg 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


34-. 




4 


P 


lg 


4- 


tr 


O 


+ 


+ 


-+- 


Strongly positive, 


44-. 




5 


M 


lg 


4- 


-+- 





+ 


+ 


-+- 


Strongly positive, 


34. 




1- 5 


M and F 


lg 


+ 


4- 





+ 


+ 


-4- 


Strongly positive, 


34. 




6 


F 


lg 


4- 


tr 





+ 


+ 


-H 


Strongly positive, 


44-. 




7 


M 


lg 


+ 


tr 





+ 


+ 


-4- 


Strongly positive, 


44-. 




8 


P 


lg 


+ 


tr 





+ 


+ 


.-+■ 


Strongly positive, 


44-. 




9 


M 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


44-. 




10 


P 


lg 


+ 








+ 


+ 


■+- 


Strongly positive, 


5-f. 




6-10 


M and P 


lg 


4- 


tr 





+ 


+ 


± 


Strongly positive, 


44-. 


6 


1 


M 


lg 


+ 


■+■ 





+ 


+ 


+ 


Strongly positive, 


34-. 




2 


F 


lg 


4- 


4- 





+ 


+ 


+ 


Strongly positive, 


34-. 




3 


M 


lg 


4 


tr 





+ 


+ 


tr 


Strongly positive, 


34-. 




4 


F 


lg 


+ 


tr 





+ 


+ 


-4- 


Strongly positive, 


44-. 




5 


M 


lg 


+ 


tr 





+ 


+ 


-+■ 


Strongly positive, 


44-. 




1- 5 


M and F 


lg 


+ 


tr 





+ 


+ 


■+- 


Strongly positive, 


44-. 







P 


lg 


4- 


tr 





+ 


+ 


-+- 


Strongly positive, 


44-. 




7 


M 


lg 


+ 


tr 





+ 


+ 


4- 


Strongly positive, 


44-. 




8 


F 


lg 


4- 








+ 


+ 


4- 


Strongly positive, 


54-. 




9 


M 


lg 


+ 








+ 


+ 


4- 


Strongly positive, 


54-. 




10 


F 


lg 


4- 








+ 


+ 


4- 


Strongly positive, 


6-r . 




6-10 


M andP 


lg 


4- 








+ 


+ 


4- 


Strongly positive, 


f> + . 


7 


1 


M 


lg 


-+■ 








+ 


+ 


4- 


Strongly positive, 


«+. 




2 


P 


lg 


4- 


tr 





+ 


+ 


4- 


Strongly positive, 


5+. 




3 


M 


lg 


4- 








+ 


+ 


Q 


Strongly positive, 


34-. 




4 


P 


lg 


-+■ 








+ 


+ 


4- 


Strongly positive, 


64-. 




5 


M 


lg 


■+■ 








+ 


+ 


4- 


Strongly positive, 


64-. 




1- 5 


M and F 


lg 


■+- 








+ 


+ 


4- 


Strongly positive, 


64-. 




6 


F 


lg 


4- 








+ 


+ 


4- 


Strongly positive, 


5-f. 




7 


M 


lg 


+ 








+ 


+ 


4- 


Strongly positive, 


54-. 




8 


F 


lg 


+ 


tr 





+ 


-\- 


4- 


Strongly positive, 


44-. 




9 


M 


lg 


4- 


tr 





+ 


+ 


4- 


Strongly positive, 


44-. 




10 


F 


lg 


+ 


tr 





+ 


+ 


4- 


Strongly positive, 


44-. 




6-10 


M and F 


lg 


+ 


tr 





+ 


+ 


4- 


Strongly positive, 


44-. 



Complements in Wassermann Reaction 



123 



TABLE 1 — Continued 
Comparison of Ten Guinea-Pig Complements 















Readings 












Number 
of 


Sex 
of 


Ambo- 
ceptor 


















Number 


Antigen 


Control 






of 


Comple- 


Guinea- 


per Tube 


Tubes 


Tubes 


Results 




Serum 


ment 


Pig 


Unit 


















1 


2 


3 


V 


2' 


V 






8 


1 


M 


lg 


+ 


tr 





J_ 


+ 


-+- 


Strongly positive, 


4+. 




2 


F 


lg 


+ 


-+- 





+ 


+ 


+ 


Strongly positive, 


4+. 




3 


M 


lg 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




4 


F 


lg 


+ 


tr 





+ 


+ 


-+- 


Strongly positive, 


4 + . 




5 


M 


lg 


+ 


tr 





+ 


+ 


± 


Strongly positive, 


4+. 




1- 5 


M and P 


lg 


+ 


tr 





T 


+ 


-+- 


Strongly positive, 


4+. 




6 


E 


lg 


+ 


tr 





T 


+ 


■+- 


Strongly positive, 


4+. 




7 


M 


1 g 


+ 


-+- 





+ 


+ 


+ 


Strongly positive, 


S+. 




8 


F 


1 g 


+ 


+ 





+ 


+ 


+ 


Moderately positiv* 


,2+. 




9 


M 


1 g 


+ 


-t- 





+ 


+ 


+ 


Strongly positive, 


3+. 




10 


P 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


4 + . 




6-10 


M andF 


lg 


+ 


± 





+ 


+ 


+ 


Strongly positive, 


3+. 


9 


1 


M 


1 g 


+ 








+ 


+ 


+ 


Strongly positive, 


5+. 




2 


P 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


5+. 




3 


M 


lg 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




4 


F 


lg 


+ 








+ 


+ 


-+- 


Strongly positive, 


5+. 




5 


M 


lg 


+ 








+ 


+ 


+ 


Strongly positive, 


6+. 




1- 5 


M and P 


1 g 


+ 








+ 


+ 


H- 


Strongly positive, 


6+. 




6 


F 


lg 


+ 


tr 





+ 


+ 


■+- 


Strongly positive, 


4+. 




7 


M 


lg 


+ 


tr 





+ 


+ 


■+- 


Strongly positive, 


4+. 




8 


P 


lg 


+ 


•¥■ 





+ 


+ 


-+- 


Strongly positive, 


3 + . 




9 


M 


lg 


+ 


tr 





+ 


+ 


-+- 


Strongly positive, 


i+. 




10 


F 


lg 


+ 








+ 


+ 


-h 


Strongly positive, 


5+. 




6-10 


M and P 


lg 


+ 








+ 


+ 


± 


Strongly positive, 


B+. 


10 


1 


M 


lg 


+ 


tr 





+ 


+ 


-+- 


Strongly positive, 


4+. 




2 


P 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


B+. 




3 


M 


lg 


+ 








+ 


+ 


tr 


Strongly positive, 


4+. 




4 


F 


1 g 


tr 








+ 


+ 


tr 


Strongly positive, 


6+. 




5 


M 


lg 


+ 








+ 


+ 


+ 


Strongly positive, 


6+. 




1- 5 


M and F 


lg 


+ 








+ 


+ 


+ 


Strongly positive, 


6+. 




6 


F 


lg 


+ 








+ 


+ 


-+- 


Strongly positive, 


6+. 




7 


M 


lg 


+ 








+ 


+ 


-+- 


Strongly positive, 


5+. 




8 


F 


lg 


+ 








+ 


+ 


tr 


Strongly positive, 


4+. 




9 


M 


lg 


+ 








+ 


+ 


± 


Strongly positive, 


5+. 




10 


F 


lg 


+ 








+ 


+ 


-4- 


Strongly positive, 


6+. 




6-10 


M and P 


lg 


± 








+ 


+ 


± 


Strongly positive, 


6+. 


11 


1 


M 


1 g 


+ 








+ 


+ 


± 


Strongly positive, 


s+. 




2 


F 


lg 


+ 








+ 


+ 


+ 


Strongly positive, 


6+. 




3 


M 


lg 


-t- 








+ 


+ 


+ 


Strongly positive, 


5+. 




4 


F 


lg 











+ 


+ 


tr 


Strongly positive, 


8-r. 




5 


M 


1 g 


tr 








+ 


+ 


-+- 


Strongly positive, 


8+. 




1- 5 


M and F 


1 g 


-+- 








+ 


+ 


-+- 


Strongly positive, 


6+. 




6 


F 


lg 


tr 








+ 


+ 


+ 


Strongly positive, 


8+. 




7 


At 


1 g 


+ 








+ 


+ 


+ 


Strongly positive, 


6+. 




8 


F 


lg 


+ 








+ 


+ 


± 


Strongly positive, 


6+. 




9 


M 


lg 


-t- 








+ 


+ 


■+■ 


Strongly positive, 


6+. 




10 


F 


lg 


tr 








+ 


+ 


-+- 


Strongly positive, 


8+. 




6-10 


M and F 


lg 


tr 








+ 


+ 


+ 


Strongly positive, 


8+. 


12 


1 


M 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


4 + . 




2 


F 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


5 + . 




3 


M 


lg 


+ 








+ 


+ 





Strongly positive, 


3 + . 




4 


F 


1 g 


+ 








+ 


+ 


tr 


Strongly positive, 


5+. 




5 


M 


lg 


+ 








+ 


+ 


+ 


Strongly positive, 


5+. 




1- 5 


M and F 


lg 


+ 








+ 


+ 


+ 


Strongly positive, 


5+. 




6 


F 


lg 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


4+. 




7 


M 


lg 


+ 


tr 





+ 


+ 


-+- 


Strongly positive, 


4+. 




8 


P 


lg 


+ 


-+- 





+ 


+ 


-+■ 


Strongly positive, 


3 + . 




9 


M 


1 g 


+ 


tr 





+ 


+ 


-+- 


Strongly positive, 


4+. 




10 


F 


lg 


-i- 








+ 


+ 


+ 


Strongly positive, 


5+. 




6-10 


M and F 


lg 


+ 








+ 


+ 


-+- 


Strongly positive, 


5+. 



124 



E. H. RUEDIGER 



Table 1 shows the reactivating power and the fixability of the 10 com- 
plements. The results obtained varied greatly. Complement 3 showed poor 
fixability and poor reactivating power. Serum 2 gave 3 -f- with Complements 
1 and 2 and 1 + with Complement 3. Poor fixability was not uniform with 
all the serums because Serum 5 fixed Complement 3 better than it did Com- 
plement 2. The mixture of 5 complements gave a fair average of the results 
obtained with the complements singly. There was no difference between 
complement from male and from female guinea-pigs. 

Test 2 

Five human complements (Complements 1-5, inclusive) were tested on 
Human Serums 7-12, inclusive. These were the same serums that were used 
in Test 1. The complements were used in doses of 0.2, 0.1 and 0.0S c c per 
test tube. Noguchi 9 recommends 0.1 c c of human complement per test tube. 



TABLE 2 
Comparison of Human Complements 













Readings 










Number 


Sex 


Ambo- 
































Number 


of 


of 


ceptor 


Antigen 


Control 




ol 


Comple- 


Guinea- 


per Tube 


Tubes 


Tubes 


Results 


Serum 


ment 


Pig 


Unit 






































1 


2 


3 


V 


2' 


3' 




7 


1 


M 


lg 


+ 


+ 





+ 


+ 


tr 


Wea ly positive, 1+. 




2 


F 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 




3 


M 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 




4 


P 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, l+. 




5 


M 


lg 


+ 


+ 


tr 


+ 


+ 


-t- 


Weakly positive, 1+. 




1- 5 


M and P 


lg 


+ 


+ 


tr 


+ 


+ 


± 


Weakly positive, l+. 


8 


1 


M 


lg 


+ 


+ 





+ 


+ 


tr 


Weakly positive, 1+. 




2 


F 


lg 


+ 


+ 


tr 


+ 


+ 


■+• 


Weakly positive, 1-*-. 




3 


M 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 




4 


F 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 




5 


M 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 




1- 5 


M and F 


lg 


+ 


+ 


tr 


+ 


+ 


± 


Weakly positive, 1+. 


9 


1 


M 


lg 


+ 


+ 





+ 


+ 


tr 


Weakly positive, 1+. 




2 


F 


lg 


+ 


+ 


tr 


+ 


+ 


■+■ 


Weakly positive, 1+. 




3 


M 


lg 


+ 


+ 


tr 


+ 


+ 


-t- 


Weakly positive, 1+. 




4 


F 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 




5 


M 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 




1- 5 


M andF 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 


10 


1 


M 


lg 


+ 


+ 





+ 


+ 


tr 


Moderately positive, 2+. 




2 


F 


lg 


+ 


+ 





+ 


+ 


tr 


Moderately positive, 2+. 




3 


M 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




4 


F 


lg 


+ 


+ 





+ 


+ 


■+- 


Moderately positive, 2+. 




5 


M 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




1- 5 


M and F 


lg 


+ 


+ 





+ 


+ 


± 


Moderately positive, 2+. 


11 


1 


M 


lg 


+ 


+ 





+ 


+ 


tr 


Moderately positive, 2+ . 




2 


P 


lg 


+ 


-H 





+ 


+ 


tr 


Moderately positive, 2+. 




3 


M 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




4 


P 


lg 


+ 


+ 





+ 


+ 


-h 


Strongly positive, 3+. 




5 


M 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




1- 5 


M and P 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2-f-. 


12 


1 


M 


lg 


+ 


+ 





+ 


+ 


tr 


Moderately positive, 2+. 




2 


F 


lg 


+ 


+ 





+ 


+ 


tr 


Weakly positive, 1+. 




3 


M 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




4 


F 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




5 


M 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




1- 5 


M and P 


lg 


+ 


+ 





+ 


+ 


■+- 


Moderately positive, 2+. 



Complements in Wassermann Reaction 



125 



The results obtained with the human complements are shown in Table 2. 
These were more uniform than those obtained with guinea-pig complement 
but were much weaker. With a mixture of 5 guinea-pig complements Serum 7 
gave 6 + and with a mixture of 5 human complements it gave 1 +. Serum 8 
gave 4 + and 1 + ; Serum 9 gave 6 + and 1 + ; Serum 10 gave 6 -f- and 2 + ; 
Serum 11 gave 6+ and 2 +, and Serum 12 gave 5+ and 2+. 



TABLE 3 
The Test As Recommended by Noguciii 



Number 


Number 


of 


oi 


Serum 


Comple- 




ment 


7 


1-5 


8 


1-5 


9 


1-5 


10 


1-5 


11 


1-5 


12 


1-5 



Sex 

ot 

Person 



M andF 
M and F 
M and F 
Mand F 
M andF 
M andF 



Ambo- 
ceptor 
per Tube 
Unit 



lg 
lg 
lg 
lg 
lg 
lg 



Readings* 



Antigen 
Tubes 



Control 

Tubes 



Results 



Negative, 

Negative, 

Negative, 

Negative, 

Weakly positive, 

Negative, 



Test 3 
Noguchi" recently advocated the use of human complement instead of 
guinea-pig complement with heated human serum. The first incubation he 
conducts in the water-bath at 37 C. for 30 minutes. In order to show how 
that method compares with our regular method Serums 7-12, inclusive, were 
tested. These serums came from patients that were known to be syphilitic. 



TABLE 4 
The Effect of Normal Human Serum on the So-Called Syphilitic Antibody 





Diluent 


Comple- 
ment 


Ambo- 
ceptor 
per Tube 
Unit 




Readings 








ol 
Serum 


Antigen 
Tubes 


Control 
Tubes 


Results 




1 


9 


3 


V 


2' 


3' 




7 


S. S. 
F. H. S. 
H. H. S. 


G-P. 
G-P. 
G-P. 


lg 
lg 

lg 


+ 
+ 
+ 


+ 
+ 
+ 




± 


+ 
+ 
+ 


+ 

+ 
+ 


-+- 

■+- 
± 


Moderately positive, 2+. 
Negative, — . 
Negative, — . 


8 


s. s. 

F. H. S. 
H. H. S. 


G-P. 
G-P. 
G-P. 


lg 

lg 
lg 


+ 
+ 
+ 


+ 
+ 

+ 


tr 
■+■ 
■± 


+ 
+ 
+ 


+ 
+ 
+ 


-+• 

± 


Weakly positive, 1+. 
Negative, — . 
Negative, — . 


8 


s. s. 

F. H. S. 
H. H. S. 


G-P. 
G-P. 
G-P. 


lg 
lg 
lg 


+ 

+ 
+ 


+ 
+ 
+ 




-+■ 


+ 
+ 
+ 


+ 

+ 
+ 


± 


Moderately positive, 2+. 
Negative, — . 
Negative, — . 


10 


s. s. 

F. H. S. 
H. H. S. 


G-P. 
G-P. 
G-P. 


lg 
lg 
lg 


+ 
+ 
+ 


-t- 
+ 
+ 




-t- 


+ 
+ 
+ 


+ 
+ 
+ 


-t- 

+ 
± 


Strongly positive, 3+. 
Negative, — . 
Negative, — . 


11 


s. s. 

F. H. S. 
H. H. S. 


G-P, 
G-P. 
G-P. 


lg 
lg 
lg 


+ 

+ 
+ 


tr 

+ 
+ 



tr 
tr 


+ 
+ 
+ 


+ 
+ 
+ 


tr 
tr 
tr 


Strongly positive, 3+. 
Negative, — . 
Negative, — . 


12 


s. s. 

F. H. S. 
H. H. S. 


G-P. 
G-P. 
G-P. 


lg 
lg 
lg 


+ 
+ 
+ 


+ 
+ 
+ 




-t- 

■+ 


+ 
+ 
+ 


+ 
+ 
+ 


■+- 

± 


Moderately positive, 2+. 
Negative, — . 
Negative, — . 



126 



E. H. RUEDIGER 



Table 3 shows the results obtained with the method recently advocated by 
Noguchi. Serums 7, 8, 9, 10 and 12 gave negative results and Serum 11 gave 
a weakly positive result, 1 +. As Table 1 shows, our regular method gave 
positive results. Serums 7, 8, 9, 10, 11 and 12 gave 6 +, 4-f, 6 +, 6+, 6 + 
and 5 +, respectively. 

Test 4 

Having found that human complement gives much weaker positive results 
than does guinea-pig complement the effect of normal human serum on syphi- 
litic human serum was studied on 6 syphilitic serums. Three portions were 
taken from each of Serums 7-12, inclusive ; one portion of each serum was 
diluted with an equal volume of salt solution containing 50% glycerol (S. S.), 
one portion was diluted with an equal volume of glycerolated fresh human 
serum (F. H. S.) and the other portion was mixed with an equal volume of 
glycerolated heated human serum (H. H. S.). At about 24 hours after these 
serums had been mixed these mixtures were tested by our regular method. 

Table 4 shows the results obtained with Serums 7, 8, 9, 10, 11 and 12 after 
they had been diluted with an equal volume of salt solution and with an 
equal volume of normal human serum. Diluted with an equal volume of salt 
solution these serums gave 2 +, 1 +, 2 +, 3 +, 3 + and 2 +, respectively, 
while all the portions diluted with an equal volume of human serum fresh or 
heated gave negative results. 

TABLE 5 
The Effeci of Guinea-Pig Serum on the So-Called Syphilitic Antibody 















Readings 










Number 
of 








Ambo- 
ceptor 
















Diluent 


Comple- 


Antigen 


Control 






Serum 






ment 


per Tube 


Tubes 


Tubes 


Results 












Unit 
























1 


2 


3 


V 


2' 


3' 






8 


S. S. 




G-P 


lg 


+ 


+ 





+ 


+ 


tr 


Moderately positive, 2+. 




G-P. S. 


1 


G-P 


lg 


+ 


+ 


-+- 


+ 


+ 


+ 


Negative, 


— . 




G-P. S. 


2 


G-P 


lg 


+ 


+ 


-H 


+ 


+ 


+ 


Negative, 


— . 




G-P. S. 


3 


G-P 


lg 


+ 


+ 


+ 


+ 


+ 


+ 


Negative, 


— ■ 


9 


s. s. 




G-P 


lg 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




G-P. S. 


1 


G-P 


lg 


+ 


+ 


+ 


+ 


+ 


± 


Negative, 


— . 




G-P. S. 


2 


G-P 


lg 


+ 


+ 


-+- 


+ 


+ 


•+■ 


Negative, 


— . 




G-P. S. 


3 


G-P 


lg 


+ 


+ 


±? 


+ 


+ 


± 


Paintly positive, 


±. 


10 


s. s. 




G-P 


lg 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




G-P. S. 


1 


G-P 


lg 


+ 


+ 


■+■ 


+ 


+ 


H- 


Negative, 


— -. 




G-P. S. 


■I 


G-P 


lg 


+ 


+ 


■+■ 


+ 


+ 


+ 


Negative, 


— . 




G-P. S. 


3 


G-P 


lg 


+ 


+ 


±? 


+ 


+ 


± 


Faintly positive, 


±. 


11 


s. s. 




G-P 


lg 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




G-P. S. 


1 


G-P 


lg 


+ 


+ 


-+- 


+ 


+ 


+ 


Negative, 


— . 




G-P. S. 


2 


G-P 


lg 


+ 


+ 


-t-? 


+ 


+ 


-)- 


Faintly positive, 


-t- 




G-P. S. 


3 


G-P 


lg 


+ 


+ 


±? 


+ 


+ 


± 


Faintly positive, 


±_. 


12 


s. s. 




G-P 


lg 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




G-P. S. 


1 


G-P 


lg 


+ 


+ 


-1- 


+ 


+ 


-+- 


Negative, 


— , 




G-P. S. 


2 


G-P 


lg 


+ 


+ 


-1- 


+ 


+ 


-+- 


Negative, 


— . 




G-P. S. 


3 


G-P 


lg 


+ 


+ 


-1- 


+ 


+ 


+ 


Negative, 


- 



Test S 

The work of Test 4 was repeated with heated guinea-pig serum (G-P. S.) 
instead of normal human serum as diluent. 

The effect of guinea-pig serum on syphilitic human serum is shown in 
Table 5. Mixed with an equal volume of salt solution Serums 8, 9, 10, 11 



Complements in Wassermann Reaction 



127 



and 12 gave 2 +, 3 +, 3 -f-, 3 + and 3 -(-, respectively, while the same serums 
mixed with equal volumes of guinea-pig serums gave negative or nearly 
negative results. 

Test 6 

The rate at which the complement-binding power disappears from syphilitic 
human serum when mixed with normal human or guinea-pig serum was 
observed on Serums 10 and 11. Three portions were taken of each serum ; 
one portion was diluted with an equal volume of salt solution (S. S.) con- 
taining 50% glycerol; one portion was diluted with an equal volume of 
glycerolated human serum (H. S.), and the other portion was mixed with 
an equal volume of glycerolated guinea-pig serum. Each portion was tested 
at the end of 2 hours, 4 hours and 6 hours. 

TABLE 6 
The Rate of Disappearance of Complement-Binding Power 













Readings* 






Number 






Ambo- 




















of 


Diluent 


Time 


ceptor 


Antigen 


Control 




Serum 






per Tube 


Tubes 


Tubes 


Results 








Unit 




































1 


2 


3 


V 


2' 


3' 




10 


S. S. 





lg 


+ 


+ 





+ 


+ 


-+- 


Moderately positive, 2+. 




H. S. 


2 


lg 


+ 


+ 


tr 


+ 


+ 


-t- 


Weakly positive, 1+. 




G.-P. S. 


2 


lg 


+ 


+ 


+? 


+ 


+ 


-+- 


Faintly positive, +. 




s. s. 


4 


lg 


+ 


+ 





+ 


+ 


-I- 


Moderately positive, 2+. 




H. S. 


4 


lg 


+ 


+ 


+ ? 


+ 


+ 


+ 


Faintly positive, ±. 




G.-P. S. 


4 


lg 


+ 


+ 


-+- 


+ 


+ 


-I- 


Negative, — . 




s. s. 


6 


lg 


+ 


+ 





+ 


+ 


+ 


Moderately positive, 2+. 




H. S. 


« 


lg 


4- 


+ 


-K' 


+ 


+ 


-+- 


Faintly positive, ±. 




G.-P. S. 


ti 


lg 


+ 


+ 


+ 


+ 


+ 


± 


Negative, — . 


11 


s. s. 


>2 


lg 


+ 


+ 





+ 


+ 


+ 


Strongly positive, 3+. 




H. S. 


2 


lg 


+ 


+ 


tr 


+ 


+ 


-+- 


Weakly positive, 1+. 




G.-P. S. 


2 


lg 


+ 


+ 


tr 


+ 


+ 


+ 


Weakly positive, 1+. 




s. s. 


4 


lg 


+ 


+ 





+ 


+ 


-+- 


Strongly positive, 3+. 




H. S. 


4 


lg 


+ 


+ 


tr 


+ 


+ 


■4- 


Weakly positive, 1+. 




G.-P. S. 


4 


lg 


+ 


+ 


+? 


+ 


+ 


+ 


Faintly positive, ±. 




s. s. 


6 


lg 


+ 


+ 





+ 


+ 


-+- 


Strongly positive, 3+. 




H. S. 


6 


lg 


+ 


+ 


+? 


+ 


+ 


-+- 


Faintly positive, ±. 




G.-P. S. 


6 


lg 


+ 


+ 


+? 


+ 


+ 


-1- 


Faintly positive, ±. 



Table 6 shows the rate at which the complement-binding power disappeared 
from Serums 10 and 11. At the end of 2 hours about 50% of the complement- 
binding power had disappeared from the portion mixed with normal human 
serum and about 75% from the portion mixed with guinea-pig serum. At 
the end of 4 hours the normal human serum had neutralized about 75% of 
Serum 10 and the guinea-pig serum had completely neutralized it. With 
Serum 11 neutralization was a trifle slower. 



Test 7 

Deterioration of guinea-pig complement was determined in the refrigerator 
ranging from 10-15 C. Three guinea-pig serums (Serums 11, 12 and 13) were 
each divided into 2 portions, A and B. Portion A was left on the clot and 
Portion B was removed from the clot and was kept in a sterile test tube. 
Each serum was tested singly and the three serums were mixed and the 
mixture was also tested. The mixture was designated X; X A was mixed 



128 



E. H. RUEDIGER 



immediately before the test while X B was mixed a few hours after the 
bleeding; the mixture was kept in the refrigerator and was tested from day 
to day. In these tests the quantity of amboceptor was kept constant. 

TABLE 7 
Deterioration of Complement in the Refrigerator 













Readings 










Number 


Portions 




Number 
































of 


A=On Clot 


Age 


ol 


Antigen 


Control 






Comple- 
ment 


B=Ofl Clot 


Days 


Serum 


Tubes 


Tubes 


Results 










1 


2 


3 


V 


2' 


3' 






11 


A 


1 


10 


-+- 








+ 


+ 


tr 


Strongly positive, 


5+. 




B 


1 


10 


+ 








+ 


+ 


tr 


Strongly positive, 


5+. 




A 


2 


10 


■+■ 








+ 


+ 


tr 


Strongly positive, 


5+. 




B 


2 


10 


+ 








+ 


+ 


tr 


Strongly positive, 


4+. 




A 


3 


10 


-t- 








+ 


+ 





Strongly positive, 


4+. 




B 


3 


10 


± 








+ 


+ 





Strongly positive, 


3+. 




A 


4 


10 


■+■ 








+ 


+ 





Strongly positive, 


4+. 




B 


4 


10 


-+- 








+ 


tr 





Moderately positive, 2+. 




A 


5 


10 


+ 








+ 


+ 





Strongly positive, 


3+. 




B 


5 


10 


tr 








+ 


tr 





Positive (Unfit). 




12 


A 


1 


10 


+ 








+ 


+ 


-+• 


Strongly positive, 


6+. 




B 


1 


10 


-+■ 








+ 


+ 


■+- 


Strongly positive, 


6+. 




A 


2 


10 


+ 








+ 


+ 


tr 


Strongly positive, 


5+. 




B 


2 


10 


+ 








+ 


+ 


tr 


Strongly positive, 


4 + . 




A 


$ 


10 


+ 








+ 


+ 


tr 


Strongly positive, 


5+. 




B 


3 


10 


■+- 








+ 


+ 





Strongly positive, 


i + . 




A 


4 


10 


■+- 








+ 


+ 


tr 


Strongly positive, 


5+. 




B 


4 


10 


■+- 








+ 


■+■ 





Strongly positive, 


3+. 




A 


5 


10 


•+- 








+ 


+ 


tr 


Strongly positive, 


5+. 




B 


5 


10 


± 








± 


tr 





Unfit 




13 


A 


1 


10 


+ 








+ 


+ 


-+■ 


Strongly positive, 


6+. 




B 


1 


10 


± 








+ 


+ 


■+- 


Strongly positive, 


6+. 




A 


2 


10 


± 








+ 


+ 


tr 


Strongly positive, 


5+. 




B 


2 


10 


+ 








+ 


+ 


tr 


Strongly positive, 


4+. 




A 


3 


10 


± 








+ 


+ 





Strongly positive, 


4+. 




B 


3 


10 


+ 








+ 


■+- 





Moderate.y positive, 2+. 




A 


4 


10 


+ 








+ 


+ 





Strongly positive, 


3+. 




B 


4 


10 


+ 


tr 





■+■ 


tr 





Unfit 






A 


5 


10 


-t- 








+ 


+ 





Strongly positive, 


3+. 




B 


5 


10 


+ 








+ 


tr 





Unfit 




X 


A 


1 


10 


+ 








+ 


+ 


■+■ 


Strongly positive, 


6+. 




B 


1 


M 


± 








+ 


+ 


-+- 


Strongly positive, 


6+. 




A 


2 


10 


± 








+ 


+ 


tr 


Strongly positive, 


5+. 




B 


2 


10 


-+■ 








+ 


+ 


tr 


Strongly positive, 


5+. 




A 


3 


10 


+ 








+ 


+ 





Strongly positive, 


4 + . 




B 


3 


10 


■+■ 








+ 


■+- 


tr 


Strongly positive, 


3+. 




A 


4 


10 


■+■ 








+ 


-H 


tr 


Strongly positive, 


3+. 




B 


4 


10 


+ 


tr 





+ 


tr 





Negative (Unfit). 






A 


5 


10 


+ 








+ 


-4- 





Strongly positive, 


3+. 




B 


5 


10 


± 








-1- 


tr 





Unfit 





The rate at which these 3 complements deteriorated in the refrigerator is 
shown in Table 7. Portion A remained fairly constant for 3 days while Por- 
tion B of the serums singly and in mixture showed considerable deterioration 
on the 3rd day. After the 3rd day deterioration of Portion A also became 
noticeable. Deterioration took place in the fixability of the complement and in 
the power to reactivate the hemolytic amboceptor. 

Test 8 
In accordance with the method described by Ramy, 2 3 fresh guinea-pig 
serums were diluted with a 10% solution of sodium acetate. A few hours 
after the guinea-pigs had been bled each serum was divided into 2 portions, 



Complements in Wassermann Reaction 



129 



A and B. Portion A was left on the clot and no preservative was added. 
Portion B was pipetted off the clot, w'as centrifuged and 4 parts of the clear 
serum were mixed with 6 parts of a 10% solution of sodium acetate in salt 
solution (9 gm. NaCl per liter). The serums were tested singly and in the 
form of a mixture of the 3 serums which is designated as X. X A was mixed 
immediately before testing and X B was mixed immediately after Portion B 
had been mixed with the sodium acetate solution. The serums were kept 
in the refrigerator and were tested at short intervals. 

TABLE 8 

Complement Preserved with Sodium Acetate 













Readings* 






Number 
ol 


Portions 
A=Not Pre- 


















Age 


Number 


Antigen 


Control 






Comple- 


served 


Days 


of 


Tubes 


Tubes 


Results 




ment 


B=Pre- 




Serum 


























served 






1 


2 


3 


V 


2' 


3' 






14 


A 


1 


10 


+ 








+ 


+ 


4- 


Strongly positive, 


64-. 




B 


1 


10 


4- 








+ 


+ 


+ 


Strongly positive, 


6+. 




B 


2 


10 


4- 








+ 


+ 


4- 


Strongly positive, 


54-. 




A 


3 


10 


4- 








4- 


+ 


4- 


Strongly positive, 


64-. 




B 


3 


10 


+ 


tr 





+ 


+ 


■+■ 


Strongly positive, 


44-. 




B 


4 


10 


+ 








+ 


4- 


tr 


Strongly positive, 


44-. 




A 


6 


10 


4- 








4- 


tr 





Unfit. 






B 


6 


10 


4- 


tr 





4- 


4- 


tr 


Positive. 






B 


8 


10 


+ 


tr 





+ 


± 


tr 


Positive. 






B 


11 


10 


+ 


tr 





+ 


4- 


tr 


Positive. 






B 


16 


10 


4- 


tr 





4- 


± 


tr 


Positive. 




15 


A 


1 


10 


4- 








4- 


+ 


4- 


Strongly positive, 


64-. 




B 


1 


10 


4- 








4- 


+ 


4- 


Strongly positive, 


64-. 




B 


2 


10 


4- 








4- 


+ 


4- 


Strongly positive, 


5$: 




A 


3 


10 


tr 








4- 


+ 


tr 


Strongly positive, 




B 


3 


10 


+ 


tr 





4- 


+ 


4- 


Strongly positive, 


44-. 




B 


4 


10 


4- 








4- 


+ 


tr 


Strongly positive, 


44-. 




A 


6 


10 


+ 


tr 





+ 


4- 


tr 


Positive. 






B 


6 


10 


+ 


4- 





4- 


+ 


4- 


Strongly positive, 


34-. 




B 


8 


10 


4- 


tr 





+ 


+ 


tr 


Strongly positive, 


34-. 




B 


11 


10 


4- 


tr 





+ 


+ 


tr 


Strongly positive, 


34-. 




B 


16 


10 


+ 


tr 





± 


± 


tr 


Positive. 




16 


A 


1 


10 


4- 








4- 


4- 


4- 


Strongly positive, 


64-. 




B 


1 


10 


-+- 








+ 


4- 


4- 


Strongly positive, 


64-. 




B 


2 


10 


4- 








4- 


4- 


4- 


Strongly positive, 


54-. 




A 


3 


10 


4- 








4- 


4- 


4- 


Strongly positive, 


64-. 




B 


3 


10 


+ 


tr 





+ 


+ 


4- 


Strongly positive, 


44-. 




B 


4 


10 


+ 


tr 





4- 


+ 


4- 


Strongly positive, 


44-. 




A 


6 


10 


4- 


4- 


tr 


+ 


+ 


4- 


Positive. 






B 


6 


10 


+ 


4- 





4- 


4- 


4- 


Strongly positive, 


34-. 




B 


8 


10 


+ 


4- 





4- 


+ 


4- 


Strongly positive, 


34-. 




B 


11 


10 


+ 


tr 





4- 


4- 


tr 


Strongly positive, 


34-. 




B 


16 


10 


+ 


tr 





+ 


± 


tr 


Positive. 




X 


A 


1 


10 


4- 








+ 


+ 


■+■ 


Strongly positive, 


6+. 




B 


1 


10 


4- 








+ 


+ 


4- 


Strongly positive, 


64-. 




B 


2 


10 


4- 








+ 


+ 


4- 


Strongly positive, 


54-. 




A 


3 


10 


4- 








+ 


+ 


4- 


Strongly positive, 


64-. 




B 


3 


10 


+ 


tr 





+ 


+ 


4- 


Strongly positive, 


44-. 




B 


4 


10 


+ 








+ 


+ 


tr 


Strongly positive, 


44-. 




A 


6 


10 


+ 


tr 





4- 


4- 


tr 


Strongly positive, 


34-. 




B 


6 


10 


+ 


4- 





+ 


4- 


4- 


Strongly positive, 


34-. 




B 


8 


10 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


34-. 




B 


11 


10 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


34-. 




B 


16 


10 


+ 


tr 





4- 


4- 


tr 


Unfit. 





Table 8 shows the results obtained by comparing complement preserved by 
Ramy's method with complement that was without preservative. On the 1st day 
after the serums had been obtained Portions A and B gave results that were 



130 



E. H. RUEDIGER 



identical. On the 3rd day Portion A of each complement and of the mixture 
X gave 6 + and Portion B gave 4 +. On the 6th day Portion A of each 
serum was practically unfit while Portion B gave 3 +. On the 8th day and 
on the 11th day all Portions B gave 3 -(- while on the 16th day all were 
unfit. In the serum mixed with sodium acetate solution complement deteri- 
oration began earlier than in the unpreserved serum but it progressed some- 
what slower. While in plain serum, Portion A, the complement showed full 
strength on the 3rd day it had lost one-third of its fixability in the preserved 
serum, Portion B. 

TABLE 9 



Glycerolated 


Complement Compared 


WITH NoNGLYCEROLATED COMPLEMENT 














Headings* 






Number 
of 


Portions 
A=On Clot 
















Age 


Number 


Antigen 


Control 






Comple- 


B=0« Clot 


Days 


of 


Tubes 


Tubes 


Results 




ment 


C=Glycer- 




Serum 






















olated 






1 


2 


3 


1' 


2' 


3' 






17 


A 


1 


10 


+ 








+ 


+ 


■+■ 


Strongly positive, 


5+. 




B 


1 


10 


+ 








+ 


+ 


-+- 


Strongly positive, 


5+. 




C 


1 


10 


-4- 








+ 


+ 


-+- 


Strongly positive, 


6+. 




A 


3 


10 


+ 


tr 





+ 


+ 


+ 


Strongly positive, 


4+. 




B 


3 


10 


+ 


tr 





+ 


+ 


-4- 


Strongly positive, 


i+. 




C 


3 


10 


-+- 








+ 


+ 


-4- 


Strongly positive, 


e+. 




A 


7 


10 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




B 


7 


10 


+ 


tr 





+ 


tr 





Negative. 






C 


7 


10 


+ 


-4- 





+ 


+ 


-4- 


Strongly positive, 


3+. 




C 


10 


10 


+ 








± 


+ 


tr 


Unfit. 




18 


A 


1 


10 


-t- 








+ 


+ 


-4- 


Strongly positive, 


6+. 




B 


1 


10 


-4- 








+ 


+ 


-4- 


Strongly positive, 


6+. 




C 


1 


10 


■± 








+ 


+ 


-4- 


Strongly positive, 


6+. 




A 


3 


10 


+ 








+ 


+ 


-4- 


Strongly positive, 


5+. 




B 


3 


10 


+ 


tr 





+ 


+ 


± 


Strongly positive, 


4+. 




C 


3 


10 


tr 








+ 


+ 


-4- 


Strongly positive, 


8+. 




A 


7 


10 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


8+. 




B 


7 


10 


+ 


tr 





+ 


tr 





Negative. 






C 


7 


10 


+ 








+ 


+ 


-+■ 


Strongly positive, 


54-. 




C 


10 


10 


tr 








■± 


+ 


tr 


Unfit. 




19 


A 


1 


10 


-+- 








+ 


+ 


-4- 


Strongly positive, 


6+. 




B 


1 


10 


± 








+ 


+ 


■+■ 


Strongly positive, 


6+. 




C 


1 


10 


-+- 








+ 


+ 


-4- 


Strongly positive, 


6+. 




A 


3 


10 


+ 








+ 


+ 


-4- 


Strongly positive, 


5+. 




B 


3 


10 


+ 


tr 





+ 


+ 


-4- 


Strongly positive, 


4+. 




C 


3 


10 


tr 








+ 


+ 


-4- 


Strongly positive, 


8+. 




A 


7 


10 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




B 


7 


10 


+ 


tr 





+ 


tr 





Negative. 






C 


7 


10 


+ 








+ 


+ 


-4- 


Strongly positive, 


5 + . 




c 


10 


10 


tr 








± 


± 


tr 


Unfit. 




X 


A 


1 


10 


-4- 








+ 


+ 


-4- 


Strongly positive, 


6+. 




B 


1 


10 


+ 








+ 


+ 


-4- 


Strongly positive, 


6+. 




C 


1 


10 


+ 








+ 


+ 


-4- 


Strongly positive, 


6+. 




A 


3 


10 


+ 








+ 


+ 


-4- 


Strongly positive, 


5+. 




B 


3 


10 


+ 


tr 





+ 


+ 


-4- 


Strongly positive, 


i+. 




C 


3 


10 


-4-? 








+ 


+ 


-4- 


Strongly positive, 


1+. 




A 


7 


10 


+ 


tr 





+ 


+ 


tr 


Strongly positive, 


3+. 




B 


7 


10 


+ 


tr 





+ 


tr 





Negative. 






C 


7 


10 


+ 








+ 


+ 


-+■ 


Strongly positive, 


5+. 




c 


10 


10 


tr 








^h 


-4- 


tr 


Unfit. 





Test 9 

Glycerol as a preservative for complement was studied on 3 guinea-pig 

serums, Complements 17, 18 and 19. A few hours after bleeding each serum 

was divided into 3 portions, A, B and C. Portion A was left on the clot, 

Portion B was pipetted off the clot and was put into a sterile test tube. 



Complements in Wassermann Reaction 131 

Portion C was pipetted off the clot, was put into a sterile tube and 1 part 
of sterilized glycerol was mixed with 3 parts of serum. All serums were 
tested singly and in a mixture composed of the 3 serums. The mixture is 
designated as X. X A was mixed immediately before testing, while X B 
and X C were prepared a few hours after the guinea-pigs had been bled. 
All complement serums were kept in the refrigerator until used, and were 
tested on the 1st, 3rd and 7th days after having been obtained from the 
guinea-pigs. 

Table 9 shows the results obtained with glycerolated complement as com- 
pared with nonglycerolated complement. Portion A showed a slight loss of 
fixability on the 3rd day, Portion B had lost a trifle more than Portion A, and 
Portion C was bound better on the 3rd day than on the 1st day. On the 
7th day Portion A was fixed or bound very poorly, Portion B was not bound 
at all, while Portion C gave results nearly equal to those obtained on the 
1st day. Portions A and B were not tested after the 7th day while Portion C 
was found to be unfit on the 10th day. 

SUMMARY AND CONCLUSIONS 

The complements of fresh guinea-pig serum varied greatly in 
fixability. While one complement gave 6 + with a certain human 
serum another complement gave only 3 -f- with the same serum, other 
things having been equal. Another serum gave 3 -j- with one comple- 
ment and 1 + with another complement. This variation in fixability 
was not due to complement alone when a complement showed poor 
fixability with a certain human serum the same complement was well 
fixed by other human serums. A mixture of 5 complement serums 
gave fairly uniform results. These results show clearly the unreli- 
ability of a single complement serum. 

Human complement gave much weaker positive results than did 
guinea-pig complement. Noguchi's recent method of doing the Was- 
sermann reaction gave negative results with serum that was known 
to be from syphilitic persons and gave 6 -j- by our regular method. 
Normal human serum and guinea-pig serum, respectively, were mixed 
with syphilitic human serum giving 6 + result with the Wassermann 
reaction; on testing them again 24 hours later negative results were 
obtained while controls which were diluted similarly with salt solution 
gave positive results. Neutralization of complement-fixing power by 
normal human serum or by guinea-pig serum progressed fairly rapidly, 
50% was neutralized in 2 hours. At the end of 4 hours neutralization 
of complement-fixing power was sometimes complete, especially by 
guinea-pig serum, which was a trifle more effective than the human 
serum. A neutralization test of this kind may become of value in 



132 E. H. Ruediger 

detecting syphilitic serum which no longer gives a positive result by 
the Wassermann reaction alone. Future observations must determine 
its value. 

Guinea-pig complement left on the clot and kept in the refrigerator 
remained fairly constant for 3 days, while complements which had 
been removed from the clots deteriorated more rapidly. Deterioration 
took place in two directions, the complement lost fixability and it lost 
the power to reactivate the hemolytic amboceptor. Complements with 
which 6 + was obtained about 24 hours after bleeding gave negative 
results with the same human serums 6 days later. Fixability dis- 
appeared more rapidly than did the power to reactivate hemolytic 
amboceptor. 

Ramy's method of preserving complement by sodium acetate was 
a total failure in my hands. Fresh guinea-pig serum mixed in pro- 
portion of 4 : 6 with a 10% solution of sodium acetate in salt solution 
began to deteriorate immediately. In 3 days it lost about 33% fixabil- 
ity while the control portion to which no preservative had been added 
remained constant. Although deterioration of preserved complement 
began earlier than of the control it progressed somewhat slower. 

Glycerol seemed to prevent deterioration of complement for a few 
days. In complement preserved by glycerol fixability increased during 
the first 3 days and on the 7th day it still was nearly equal to the 
fresh serum.