658 



COLLECTED STUDIES IN IMMUNITY. 



have been anchored, and then, owing to the secondary tightening 

 of the bonds, would no longer have been available even for a group 

 possessing somewhat higher affinity. One can easily convince one's 

 self of the part played in deflection by the sequence in which the 

 various reagents are added. In a recent study, Michaelis and 

 Fleischmann 1 have called attention to the sources of error to which 

 disregard of this circumstance may give rise. 



We made the following experiment with our antiserum : 



Two series of tubes were prepared, each containing 0.1 cc. antiserum and 

 decreasing amounts of normal rabbit serum. The volume was made up to 

 l.occ. and the mixtures allowed to stand for twenty-four hours in order to 

 secure the maximum amount of precipitation. The tubes were sharply cen- 

 trifuged, and the supernatant fluid removed. To the sediments in series A 

 were then added 0.075 cc. guinea-pig serum, and the mixtures kept at 37 for 

 one hour. Then the ox blood, plus 0.0015 cc. rabbit amboceptor, was added. 

 In series B, the sequence was altered to: ox blood, plus 0.0015 cc. amboceptor 

 one hour at 37 then 0.75 cc. guinea-pig serum. 



The degree of haemolysis at the end of 1J hours is shown in Table IV. 



TABLE IV. 



It will be seen that the precipitate has exerted a deflection of 

 complement, though not to a very high degree; there is no deflection, 

 however, when the sequence in which the various reagents are added 

 is the same as that employed in our antiamboceptor experiments. 2 



The essential importance of the technique employed, when 



1 Michaelis and Fleischmann, Zeitsch. f. klin. Medizin. Vol. 58, 1906. 



2 It is impossible for us to say whether the sequences in which the reagents 

 are added would have the same determining influence when other ambo- 

 ceptors, especially bactericidal amboceptors, are employed. 



