44 



3rd Ralibit— 1 ce. norm. rabb. ser.— 

 0,1 Shiga extract. 



4th Rabbit — 0,1 Shiga s. — 0,4 Shi- 

 ga extract. 



The first rabbit died within three 

 days with generalised paralysis, the se- 

 cond one, whicla had shown itself to be 

 almost entirely paralytic from the second 

 day on, resisted until the ninth day. 

 The remaining two animals showed no 

 no alteration of their state of health. 



Upon repeating these experiments we 

 obtained similar results. The first rabbit 

 died on the 3rd day, the second on the 

 7th, with initial paralysis on the 2nd. The 

 precipitating serum of 4 days, used in 

 this case, had proved itself to be entirely 

 incapable of removing the tetanus anti- 

 toxin from the antitoxic serum. Extending 

 our investigations to the agglutinins, we 

 employed not only the technic of KRAUS 

 and P11ÍBRAM, starting from initial dilu- 

 tions to make the successive dilutions, 

 but using also for every dilution to 

 which germs had to be added, 0,5 of 

 precipitating serum. 



Technic. 



To each tube containing 0,5 of pre- 

 cipitating serum were added 0,5 of dilu- 

 tions at 1/25, 1/125, 1/250. 1/500, 1/1000, 

 1/2000, 1/4000 of dysentery agglutinating 

 serum and afler two hours in the incu- 

 bator at 37". Centigrade, the precipitates 

 formed were removed by centrifugation 

 and- décantation. Placing now in each 

 lube 1 cc. of an emulsion of Shiga bac, 

 the mixture was placed in the incubator 

 for another two hours and then results 

 were read. 



We made several series for the pre- 

 cipitins we had, of 4, 10, 16. 21. and 26 

 ■days. 



Agglutination was positive up to 

 1/16000 (last tube) in the series in which 

 4ih day precipitins were used and com- 

 pletely negative in all the others, right 

 from the first tube, although the dilution 

 of the serum employed did not exceed 

 1/25. 



A control series showed that the se- 

 rum was agglutinating up to 1/500. 



The agglutination up to 1/lGOOO in 

 the series of precipitins of the 4th day 

 was due to the rabbit serum used. 



For typhus agglutinins the serum em- 

 ployed was agglutinating for the sample 

 used up to 1/10.000. 



.\s before for dysentery bacilli the 

 agglutination was negative with precipi- 

 tins of 10th, I6lh. 21th, and 26th days 

 and positive with the one of the fom'th 

 day. Besides the technic indicated above, 

 we made series of dilutions with aggluti- 

 nating serum at 1/500 and 1/1000 in 0.5 

 cc. which we added to 0,5 of the precipi- 

 tating sera. Afler the lapse of time indi- 

 cated for remaining in the incubator and 

 after removing the precipitate, we took 

 the remaining liquid for making dilu- 

 tions whicli we mixed with emulsions of 

 typhus bacilli permitted up to obtain li- 

 tres oí 1/1000, 1/2000, 1/3000, 1/8000, 

 1/16.000, 1/32.000. 1/64.000. 



The reading permitted here also 

 with the precipitins of the 4th day posi- 

 tive agglutination up to 1/32.000; this 

 was probably owing to the rabbit senun. 



These indications show that diffe- 

 rent antibodies are to be found in cir- 

 culation as parts of differenl precipiti- 

 nogens, which can be partially distin- 

 guished by the delay with which the pro- 

 duction of precipitins takes place in the 

 organism of the rabbit. Thus the dysen- 

 tery antitoxin is already partially preci- 

 pitated by precipitins of the foiu*th day, 

 whilst agglutinins for the same genn are 

 only precipitated by sera ,of the tenth 

 day and the antitoxin is only removed 

 from antitoxic serum by sera older still 

 (16 days). 



For the two agglutinins we investi- 

 gated, the precipitation began with sera 

 of the same date, whereas with the diph- 

 theria and bothropic antitoxins it was 

 always impossible to obtain positive re- 

 sults, in spite of all the trouble we took 



