The measurement of the combining power of diphtheria toxin and toxoid with anti-toxin in relation to their antigenic efficacy / by A.T. Glenny, C.G. Pope, and Hilda Waddington.

Glenny, A. T.

Date:: [1925?]

Credit: The measurement of the combining power of diphtheria toxin and toxoid with anti-toxin in relation to their antigenic efficacy / by A.T. Glenny, C.G. Pope, and Hilda Waddington. Source: Wellcome Collection.

11/28 (page 287)

$that at the end of 30 minutes’ exposure to a temperature of 50° C. marked flocculation occurred in the mixture containing 0'155 c.c. of toxin to each unit of antitoxin and was beginning in the tube on either side containing O'160 and 0‘150 c.c. of the toxin; traces of precipitate could be seen forming in the tube containing 0’145 of the toxin. No other tube showed any signs of precipitate formation ; the Lf value therefore of this toxin is taken as 0*155 c.c. Mixtures containing toxin increasing from 0*175 c.c. to 0*22 c.c. with 1 unit of antitoxin were injected subcutaneously into guinea-pigs and it was found that the mixtures containing 0*21 c.c. of toxin killed on the fifth day, while mixtures with more toxin killed earlier and those with less caused a late death or failed to kill. The L-f- value of the toxin is therefore 0*21 c.c. In the same series of animals injected subcutaneously those receiving more than 0*18 c.c. of toxin with 1 unit of antitoxin showed local oedema, those injected with 0*18 c.c. or less gave no signs of swelling. The Lo value of the toxin is therefore 0*18 c.c. The mixtures containing from 0*16 to 0*185 c.c. of toxin were injected intracutan- eously into guinea-pigs in doses of 0*2 c.c.; the Lo mixture containing 0*18 c.c. of toxin produced a very marked inflammatory reaction and the mixture contain¬ ing 0*175 is taken as the Lr dose, because a small reaction was produced by this mixture and no reaction by mixtures containing less than this quantity of toxin. The mixture containing 0*20 c.c. of toxin when diluted 1 in 500 gave an intra- cutaneous reaction which was not caused by the injection of the same dilution of the mixtures containing less toxin j the Lr/500 dose of this toxin is therefore l/500th of 0*20 c.c., or 0*0004 c.c. We may point out here that the Lr dose is not exactly 500 times the Lr/500 because, as we have already mentioned, the Lr/500 test is less sensitive than the Lr and therefore gives a lower neutralisa¬ tion level to the toxin. We have not included this titration in table I. because we do not regard the Lr/500 dose as a definite unit j other fractions are often used by us for convenience of experiment and these fractional tests must be regarded as rough estimations or as preliminary determinations to avoid the danger of killing guinea-pigs by excess of free toxin which might result if titrations for the full Lr dose were made upon toxins of unknown strength. Before considering the theoretical relation of these various measures one to another, and their connection with antigenic value we record a series of experiments establishing certain relationships between the toxin and toxoid content of a toxin brew, its combining power and its antigenic value. Experimental results. The ratio hetiveen Lf and IjT doses is not constant. If flocculation usually occurs first in mixtures over-neutralised according to animal test, it follows that toxins appear slightly stronger in combining power when tested in vitro than when tested in vivo, and the Lf dose is less than the Lo or Lr dose. The ratio Lf/Lr in the example quoted in table 1. is 0*155/0*175 i.e. 0*88, and in the majority of fresh batches of toxin tested against our standard anti¬ toxin has been from 0*80 to 0*95. If flocculation indicates an exact balance between antigen and antibody this ratio indicates that no inflammatory reaction is produced when a guinea-pig is injected intracutaneously with toxin of which from 80 per cent, to 95 per cent, has been neutralised by antitoxin. If, on the other hand, the Lr A4$