STANHOPE BAYNE-JONES 763 



cultures of toxin-producing bacteria. The non-toxic antigenic portion of the filtrate 

 adds a proportional combining capacity to the whole mixture. This proportion may 

 vary from the condition in a fresh toxin where there is little toxoid to that in an old or 

 formalinized product, completely devoid of toxicity while retaining its original floccu- 

 lating value. Hence, there is no necessary relationship between the M.L.D., Lo, L+, 

 Lf, and other doses of toxin. The case of a fresh toxin, however, is a special one which 

 demonstrates that there is a fundamentally close relationship between toxicity and 

 the flocculating value. As the Lj value is the amount of toxin equivalent to i unit of 

 antitoxin, a ratio of Lo/i,/= i should be obtained. Such a unit ratio, however, is rarely 

 found. The Z/ values are often twice or three times as great as the Lo values determined 

 by animal tests. Nevertheless, Ramon (1922-23), Scholz (1925), and Glenny and 

 Wallace (1925) have occasionally found Lo/Lf ratios equal to i with certain toxins 

 and selected antitoxic sera. It seems to be advisable, at present, to follow the example 

 of Glenny and Wallace (1925) and use such provisional terms as "iw vitro unit" and 

 "in vivo unit," until the significance of the differences between the results of animal 

 tests and flocculating values has become fully understood. As the measurements 

 made by animal tests indicate only partial neutralization of a toxin, the flocculation 

 method is the only true measure of the combining capacity of a toxin. It has an in- 

 dependent meaning, and can be used to establish an almost self-contained system. 



NATURE OF THE REACTION 



Two zones of flocculation occasionally occur in mixtures of toxin and antitoxin 

 and discrepancies as great as 300 per cent between flocculation titrations and animal 

 tests have been observed. These results have cast doubt upon the opinion that the 

 flocculation is a consequence of a specific reaction between toxin and antitoxin. Wein- 

 berg, Prevot, and Goy (1924), Zingher (1924), Moloney and Weld (1925), Bronfen- 

 brenner and Reichert (1926), and Eisler and Kovacs (1926) have drawn attention to 

 the agglutinins and precipitins which occur in antitoxic sera, and have again raised 

 the question whether the flocculation reaction is only the long-familiar bacterial pre- 

 cipitin phenomenon. The generally close agreement between the results of animal 

 tests and flocculation reactions which have been obtained by many workers in thou- 

 sands of titrations of diphtheria toxins and antitoxins, and with other toxins and anti- 

 toxins, which will be set forth in sections dealing with these special cases, seems to in- 

 dicate that there is more than a fortuitous connection between the in vivo and in vitro 

 results. The uniformity of the method of making diphtheria toxin throughout the 

 world and the constant use of the Park No. 8 strain of B. diphtheriae are not sufficient- 

 ly uniform methods to balance the differences in the culture media and the differences 

 in horses under immunization. It is easily demonstrated, however, that an antibac- 

 terial serum will precipitate toxic filtrates, and that some antitoxins will agglutinate 

 bacteria and cause precipitates in solutions of bacterial protein which were never spe- 

 cifically toxic. Further examination of these conditions discloses the following: 



There is no relationship between the agglutinating titre of an antitoxic serum and 

 its flocculating value when mixed with a toxin. Moloney and Weld (1925), misquoted 

 by Bronfenbrenner and Reichert (1926), found that there was "no obvious relation 

 between the agglutinin titre of a serum and its antitoxic content." Similar results 



