Gay and Rusk.] THE LOCUS OF ANTIBODY FORMATION. 7 



method to the conversion of glycogen to glucose, and at this point 

 introducing Betrand's modification of Fehling's method as more 

 accurate in measuring the amount of copper reduced. It is found 

 that the microchemical method serves to give somewhat definite in- 

 formation as to the amount of glycogen present, but within a limited 

 range only, for when the chemical analysis showed very much or 

 very little glycogen the staining method was at times wholly 

 inadequate. 



III. THE FATE OF HORSE SERUM INJECTED INTRAVENOUSLY IN NORMAL 

 AND IMMUNIZED RABBITS. 



The two general methods that have been and may be employed in 

 seeking antibody origin are either to trace the course of the injected 

 antigen to some group of cells or to seek the precocious appearance 

 of antibodies in extracts of a given group of cells. The latter method 

 is the one that has been used most frequently, but it is the former 

 that we have employed. Our observations, some of which have 

 already been published, 42 began with a study of the fate of horse 

 serum injected into the blood stream of rabbits that had been im- 

 munized against horse serum. They have since led to further studies 

 on the result of an initial injection of horse serum in normal rabbits. 

 In all instances our results deal with an injection of one cubic centi- 

 meter of serum intravenously. 



In beginning the experiments with immunized animals it was neces- 

 sary first to determine the best method of detecting the antigen that 

 was reinjected. It was found that when horse serum is injected intra- 

 venously in rabbits that have a high precipitin content for horse serum 

 it nevertheless remains demonstrable by the fixation reaction or the 

 precipitin reaction for 24 hours. The reactions are carried out by 

 adding the antigen-containing antiserum to a pure antiserum. After 

 48 hours the antigen is no longer demonstrable. The persistence of 

 the antigen in the immune animal is accompanied by a fall in the 

 precipitin value of serum (negative phase). It is of interest to note 

 that although this antigen-containing antiserum will not precipitate 

 or fix alexin spontaneously, it will react with another antigenic anti- 

 serum as well as with a pure antiserum. It was rather surprising to 

 us to fail in any conclusive demonstration of the antigen by the 

 fixation reaction in extracts of the organs of these same immunized 

 animals (spleen, lymph nodes, liver, kidney, and muscles) either 

 at the same time the antigen is present in the blood or even 24 hours 

 later. 



Of undoubtedly greater significance is the fact that neither the 

 antigen-containing antiserum, nor the organ extracts of the same 

 animal will sensitize guinea pigs to subsequent intoxication by horse 



