Comparative studies of the in vivo toxin neutralization and the in vitro Vero cell assay methods for use in potency testing of diphtheria component in combined vaccines/toxoids. 1: Standardization of a modified Vero cell assay for toxin-antitoxin titration of immunized guinea-pig sera.

The in vivo Toxin Neutralization (TN) test is currently used as an antibody limit test for regulatory control testing of diphtheria potency in combined vaccines/toxoids. This test requires the use of guinea-pigs for estimating diphtheria antitoxin titre in immunized sera. The acceptability criteria (minimum requirement) for adsorbed pediatric vaccines/toxoids in the TN test are the production of at least 2.0 IU/ml of antitoxin in the serum pool of immunized animals. The feasibility of employing the in vitro Vero cell assay as an alternative limit test to the TN test was investigated. Parallel titration of serum samples from guinea-pigs immunized with DPT (ads) vaccine (pediatric use) showed that although the in vitro antitoxin titre paralleled the in vivo values, a direct correlation between the two methods could not be established. The mean in vitro titres were lower (in terms of IU/ml) than those obtained by the in vivo TN test. This could possibly be explained by the differences between reference serum which is a hyperimmune horse serum and the test sera which are from guinea-pigs which have been immunized only once. The in vitro/in vivo ratio of the antitoxin titre ranged from 0.1 to 0.24 IU/ml with a mean value of 0.17 (SD, 0.06). Hence, for pediatric vaccines, a tentative in vitro limit of 0.3 IU/ml, which would correspond to the currently required limit of 2.0 IU/ml in the in vivo TN test, could be accepted as the minimum requirement for the antitoxin titre in the Vero cell assay.

Cell-free ammonia-oxidizing system of Nitrosomonas europaea: general conditions and properties.

Conditions essential for the preparation of active ammonia-oxidizing extracts of Nitrosomonas europaea were studied. The extracts were unstable during storage and required specific assay conditions for ammonia oxidation. Bovine serum albumin, spermine, or MgCl2 was required for ammonia oxidation and the concentration of phosphate determined the relative effectiveness of each activator, i.e., albumin being most effective in 0.1 M phosphate and spermine or MgCl2 at lower phosphate concentrations. Kinetic experiments showed a partial reduction of cytochrome c preceding the initiation of oxygen consumption due to ammonia oxidation.

Surface membrane changes of T cells induced by syngeneic tumour cells. II. T-cell defects induced by small tumour cell inocula or tumour cell antigens.

Injection of a large number of tumour cells, like other strong immunogenic challenges, is followed within 6 h by the uptake of cytophilic Ig (probably complexes) by a subpopulation of T cells. This phenomenon, known as the "6-hour T-cell response" is abrogated when small tumour cell inocula (10(2)), or small amounts of a preparation from tumour cells, which contains tumour antigens, are injected prior to the immunogenic challenge Abrogation of the "6-hour T-cell response" resulted in a decrease in specific anti-tumour cell immunity as tested in vitro by measuring growth inhibition (cytostasis). It has also resulted in loss of the amplifying function on antibody formation against sheep erythrocytes, normally detected in a T-B cell co-operative system when T cells are used 6 h after priming with sheep erythrocytes. It is postulated that this T-cell defect may represent a mechanism by which tumour cells, in the early stages of their growth, interfere with inductive stages of the immune response for a sufficient period of time to allow the tumour to grow beyond immune control.

Surface membrane changes of T cells induced by syngeneic tumour cells. I. Formation and uptake of complexes of Ig and tumour antigens by T cells.

A large dose of tumour cells (10(7)) from two different tumours produced a significant increase of Ig-bearing spleen cells 6 h after intraperitoneal administration in syngeneic mice, like conventional antigens previously tested. Incubation of normal spleen cells in serum taken 6 h after administration of tumour cells reproduced in vitro the changes observed in the spleen cell population of the serum donors, demonstrating the presence of a cytophilic Ig, which was shown to be taken up by T cells. Serum collected 6 h after tumour cell inoculation contains also a 4S factor which can generate in vitro cytophilic Ig for T cells in the presence of a foreign soluble protein or tumour antigen. Extracts obtained from tumours cells, which were shown to contain tumour antigens, were labelled with 125I and mixed with the 4S factor and normal 7S Ig. Upon fractionation of the mixture, high molecular weight material containing radioactivity and 7S Ig were eluted in the void volume, well ahead of the position of both the tumour cell preparation and the 7S Ig. This material contained Ig cytophilic for splenic T cells and it is likely that it represents cytophilic complexes of Ig and tumour antigens. It is postulated that the cytophilic Ig detected in the serum of animals 6 h after injection of a large number of tumour cells represents cytophilic complexes of Ig and tumour antigens formed through the mediation of a soluble factor.

Ammonia or ammonium ion as substrate for oxidation by Nitrosomonas europaea cells and extracts.

The effect of pH on the K(m) values for ammonia was studied in its oxidation by Nitrosomonas cells and cell-free extracts. The K(m) values decreased markedly with increasing pH, suggesting (NH(3)) rather than (NH(4) (+)) as the actual substrate for oxidation.