The stability and solubility of diphtheria toxin in acid and alkili / Una Wallace.

  • Wallace, U.
Date:
[1927?]
Catalogue details

Licence: In copyright

Credit: The stability and solubility of diphtheria toxin in acid and alkili / Una Wallace. Source: Wellcome Collection.

    2/8 page 668
    Outline of experiments. In a previous publication it was shown that the loss in yield of toxin throughout a series of toxins precipitated with acid was never less than 40 per cent. (Watson and Wallace 19246). This may have been due to any or all of the following causes destruction ; loss in handling the slight precipitate ; incomplete precipitation of the toxins; under-estimation of the purified toxin by measuring the Lr/500 dose, since the toxins may have been partially “ toxoided” during the process (Doerr 1908). These points have now been investigated separately. The destruction is traced from^H 0*5 toj^H 11-0 in a series of controls in which loss in handling the precipitate is eliminated; the partition of the toxin between the clear solution and the precipitate is estimated over the three separate ranges of ydH where precipitation occurs; the value of the toxin is determined by Ramon’s flocculation method which measures the sum of the toxin and toxoid present. Samples of the toxin were brought to various hydrogen ion concentrations with HC1 and NaOH of such strengths as to make the change in volume negligible in the estimation of the toxins i.e. the maximum increase was 5 per cent. After two days at room temperature, the limits of precipitation were noted: the whole of the clear samples, and one half of those which had precipitated, were then readjusted to the pH of the original toxin. The remaining half of each of the precipitated samples was centrifuged: the supernatants separated, and readjusted to pH 8-0 after filtering. The precipitates were washed with the original volume of diluted buffer solution of the same pH as the solution from which they were separated. They were finally dissolved with a trace of NaOH and diluted to the original volume at pH 8’0. This routine was first carried out with the original toxin containing 0'2 per cent, formalin, then with the same toxin to which 0’5 per cent, phenol had been added. Later, with the object of a more complete precipitation of the toxin, 2 per cent, alum was added, and to investigate the action of the alum it was added in the presence of tartrate (charts 1-4). Measurement of the toxin. Toxin is characterised by three different effects : toxicity, combination with antitoxin and antigenic power; each has given rise to separate methods of measurement and until all three effects have disappeared the toxin cannot be said to have been destroyed. The original toxin was already partially toxoided, therefore no attempt was made to measure further changes by toxicity. Combination with antitoxin was measured in terms of Lf units per c.c. by titration with standard serum as described by Glenny and Wallace (1925), and antigenic values by the comparative method of the immunity index (Glenny, Allen and Hopkins, 1923). The flocculation test may give erroneous results when applied to toxins concentrated by acid precipitation. Other substances occurring in the culture filtrate may be simultaneously concentrated and give non-specific zone flocculations. Such flocculations may or may not appear with normal horse serum and can only be distinguished by titrating against several different standard sera, when the non-specific flocculation, should it again appear, will be seen to bear no constant relation to the antitoxic content of the various sera. One such instance has been published by Glenny and Wallace (1925), and since several others have subsequently occurred it is advisable to check the Lf value of treated toxins by some animal test. Toxins which have been treated with acid or alkali may give no flocculation with antitoxin. It does not follow that they have been destroyed, for their Lf value may still be measured by blending with fresh toxin. In these experiments the original toxin was
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