Guinea-pigs given two doses of 2–25 Lf of fluid tetanus toxoid at 28 days interval had very satisfactory antitoxin titres 10 days after the second dose of toxoid (g.m. 28–2 units/ml.). Similar groups of animals given 150 units of horse tetanus antitoxin simultaneously with the first dose of toxoid responded very badly (g.m. < 0·016).
Interference by passive antitoxin occurred even when the antitoxin was given as late as 4 days after the first dose of toxoid.
Interference by passively administered antitoxin was minimal when aluminium hydroxide-adsorbed toxoid was used. It was necessary to increase the dose of antitoxin from 150 to 2400 units before significant interference occurred.
The route of administration of antitoxin did not significantly affect the results except when the antitoxin was given intravenously.
When guinea-pigs were immunized and bled at regular intervals it was found that with both fluid and aluminium hydroxide-adsorbed preparations, titratable antitoxin was present on the 14th day. The increase in titre thereafter was more rapid with the adsorbed preparation, but after a second dose of toxoid there was no significant difference in titre.
Passively administered antitoxin virtually abolished the active response to fluid toxoid, but with aluminium hydroxide-adsorbed preparations the primary response was not abolished but reduced and delayed and there was much individual variation.
Horse serum-sensitive guinea-pigs given adsorbed toxoid with simultaneous passive horse antitoxin gave a better primary response to the toxoid than did unsensitive animals.
The effectiveness of adsorbed tetanus toxoid in the simultaneous immunization procedure was directly related to the concentration of aluminium hydroxide or phosphate used: this concentration was critical and amounts below a certain level were ineffective. Calcium phosphate used as an adsorbent was unsatisfactory in this way, although it was an excellent adsorbent.
Investigation of the adsorbent characteristics of aluminium hydroxide and phosphate and of calcium phosphate, showed that the calcium salt on a molar basis was the most effective and that aluminium phosphate was the least effective.
Elution of toxoid from centrifuged precipitates of the three types of adsorbent showed that only 5% of toxoid was removed from the aluminium hydroxide, 13–18% from the calcium phosphate and 31–33% from the aluminium phosphate preparation when incubated with normal serum at 37° C.
Aluminium hydroxide adsorption in vitro interfered with the ability of antitoxin to combine with toxoid and to a lesser extent calcium phosphate had the same effect; aluminium phosphate, however, did not appear to interfere at all in this way
Histological observations on the tissue response to aluminium phosphate and calcium phosphate indicated that the typical alum granuloma produced by aluminium phosphate was not produced by the calcium salt.