The processing and collaborative assay of a reference endotoxin.

A preparation of Escherichia coli bacterial endotoxin, the latest of successive lots drawn from bulk material which has been studied in laboratory tests and in animals and humans for suitability as a reference endotoxin, has been filled and lyophilized in a large number of vials. Details of its characterization, including stability studies, are given. A collaborative assay was conducted by 14 laboratories using gelation end-points with Limulus amebocyte lysates. Approximate continuity of the unit of potency with the existing national unit was achieved. The lot was made from the single final bulk but had to be freeze-dried in five sublimators. An assessment was therefore made for possible heterogeneity. The results indicate that the lot can be used as a large homogeneous quantity. The advantages of using it widely as a standard for endotoxins are discussed.

The second international standard for polymyxin B.

Since supplies of the first International Standard for Polymyxin B were exhausted, it was replaced by a second international standard the potency of which was estimated from the results of a collaborative assay carried out by 5 laboratories in 4 countries. The wide variations in the results probably resulted from difficulties experienced in handling the first international standard. The potency finally agreed upon by the collaborating laboratories, on the basis of the overall mean values obtained after rejection of the most discrepant assays, was 8 403 IU/mg. That value was accepted by the WHO Expert Committee on Biological Standardization (1970), which consequently defined the International Unit of polymyxin B as the activity contained in 0.000119 mg of the second international standard.

International collaborative studies on potency assays of diphtheria and tetanus toxoids.

Collaborative studies showed that relative potency assays for a particular type of diphtheria toxoid (adsorbed) and for tetanus toxoid (plain and adsorbed) gave very similar results, whether the assays were carried out by toxin challenge or by antitoxin titration after immunization of experimental animals with graded doses of toxoid. The same numerical results were obtained with a scoring system as with a system based on survivals only. Although skin tests were used on a very limited scale in these studies, it seems likely that they could replace lethal tests for the diphtheria challenge assays.For both tetanus and diphtheria, the adsorbed toxoid gave a higher relative potency when combined with other antigens than as a single toxoid. Both mice and guinea-pigs were used for the lethal challenge test of adsorbed tetanus toxoid. For the single tetanus toxoid the results were the same, but for the combined toxoid (DPT vaccine) the mouse assay results were about twice those of guinea-pig assays.