752 TOXINS, TOXOIDS, AND ANTITOXINS 



toxified with formalin is precipitated by adding i per cent glacial acetic acid. The 

 precipitate is allowed to settle. The clear supernatant fluid is siphoned off; the re- 

 mainder, centrifugated. The clear supernatant is again siphoned off and replaced by 

 an equal volume of 0.7 per cent saline or sodium acetate — acetic acid mixture, ad- 

 justed to the pH of the acid precipitation-point (ca. 3.8) to wash the precipitate and 

 again centrifugated. The precipitate is dissolved in physiological saline or distilled 

 water buffered to pH 8.0, using the volume needed to give the desired concentration. 

 They state that the purification of the toxoids can be carried out at room temperature. 

 That there is some loss through absorption on filtering through a Berkefeld is shown 

 by the flocculation value. 



The ultra-purification of the purified toxoids (Moloney and Weld)' can be ac- 

 complished by adding alcohol to give an 85 per cent final concentration or by the 

 dialysis of the solution for seventy-two hours against water. Purified toxoids, they 

 state, are protein-like substances and give a positive biuret, Millon, xanthoproteic, 

 and Hopkins-Cole tests. 



Nicolle, Debains, and Cesari- and Ramon^ described the formation of a precipi- 

 tate when toxin is neutralized with antitoxin, and Nicolle recommended this as a 

 means of determining the unit value of antitoxin. The writer^ precipitated two differ- 

 ent toxin preparations (Nos. 377 and 517) by adding antitoxin to under the neutraliz- 

 ing-point. The toxin-antitoxin precipitate was collected by centrifu gating and was 

 then redissolved in saline equal to half the original volume. This was slightly toxic. 

 One-mil doses of the preparation from toxin No. 377 in 250-gm. guinea pigs caused 

 late paralysis and death in twenty-four to thirty days. One-mil doses of the prepara- 

 tion from toxin of No. 517 caused slight paralysis in some of the guinea pigs with 

 recovery. Both of these precipitated toxin-antitoxin preparations gave a higher per- 

 centage of immunity in guinea pigs than the o.i L+ to.xin-antitoxin preparations 

 and the toxoid preparations used for comparison. Hartley^ obtained similar results 

 in his investigations of the antigenic properties of precipitates produced by the inter- 

 action of diphtheria toxin and antitoxin. He prepared overneutraUzed, neutral, and 

 underneutralized (toxic) mixtures, and he obtained from the toxic mixtures precipi- 

 tates containing only one-five-hundredth (0.2 per cent) of the total nitrogen of the 

 original mixture and still these precipitates were very active immunizing agents. 

 Doses containing less than a ten-thousandth of a milligram of nitrogen stimulated the 

 production in six weeks from 0.02 to o.i units of antitoxin in guinea pigs. As stated 

 earlier, the purest toxoid obtained by Watson and Wallace was two hundred times^ 

 purer than the original toxin from which it was prepared. This is less purification 

 than Hartley obtained. 



'Moloney, P. J., and Weld, C. B.: loc. cit. 



' Nicolle, M., Debains, E., and Cesari, E.: Compt. rend. Acad, dc sc, 169, 1433. iQig; Ann. dc 

 rinst. Pasteur, 34, 709. 1Q20. 



J Ramon, G.: Ann. de Vlnst. Pasteur, 37, loor. 1920; 38, 1. 1924. 



"Park, \V. H., Banzhaf, E. J., Zingher, A., and Schroeder, M. C: Am. J. Pub. Health, 14, 

 1049. 1924. 



5 Hartley, P.: Brit. J. Exper. Path., 6, 112. 1925; 7, 55. 1926. 



'' Cf. Glenny, A. T., Hopkins, B. E., and Pope, C. G.: /. Path, b- Bact., 27, 261 n. 1924. 



