Ionic interfaces and diphtheria toxoid interactions

We present here a systematic study on the purification of the diphtheria toxoid (Dtxd) produced at the Instituto Butantan, by adding only one step on the entire process of its production. Aliquots of 1.0 ml of Dtxd were added to an equal amount of Q-Sepharose previously equilibrated with 500 mM Tris, pH 5.0-9.0 (increments of 0.5 pH units). The best condition for the Dtxd monomer adsorption was achieved at pH 9.0. The best condition for desorption was obtained with 300 mM NaCl. After studying the gel binding capacity for Dtxd, a column (C20/20) equilibrated with 500 mM Tris, pH 9.0, was prepared. The purification factor for Dtxd was 1.5. The final recovery of Dtxd was 68.75%, with 90.31% purity. The process methodology presented here is a very realistic sequence of separation steps, which is perfectly compatible with the production requirements. Vaccination with "toxoid highly purified toxin" is known to confer a strong immunity on people in the absence of undesirable reactions, which led experts of European Pharmacopoeia to recommend its use both for children and adult vaccination.     

In vitro induction of a diphtheria toxoid specific antibody response in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid

In comparison with the presently used potency test for diphtheria vaccine, in vitro examination of the immunogenicity of the vaccine would have great advantages. For this reason in vitro induction of diphtheria toxoid specific antibody synthesis in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid was investigated. The results showed that a dose dependent synthesis of diphtheria antibody was induced by adsorbed diphtheria toxoid and combined vaccines containing the diphtheria toxoid component. Plain diphtheria toxoid appeared to be less immunogenic in comparison with adsorbed toxoid. There is some indication that the pertussis component had a stimulating effect on the diphtheria antibody synthesis. In conclusion, these results are promising for in vitro examination of the immunogenicity of diphtheria vaccines. The model will be validated for the routine control of diphtheria vaccine.     

Hemocytoblastoid reaction of thymic subcapsular cells to diphtheria toxoid

Subcapsular cells lining the thymic stroma vary from low to high forms, while others have a hemocytoblastoid aspect. The purpose of the present study was to establish whether the transformation of the low forms into hemocytoblastoid subcapsular cells can be induced by an antigen. Rats given 10 Lf of diphtheria toxoid intramediastinally were killed at periods ranging from 3 to 24 hours later. Other rats were injected with ³H-thymidine at various intervals after the toxoid injection, and were killed one hour later. The observations revealed a rapid hemocytoblastoid transformation of subcapsular cells following administration of the toxoid. The transformation is detectable as early as three hours after the injection and can be completed after nine hours. Radioautography revealed that DNA duplication is initiated rapidly in the transforming subcapsular cells, since it can be completed 9 to 12 hours after the toxoid injection. Other observations suggested the transformation of reactive perithymic fibroblasts into subcapsular cells as well as the transformation of hemocytoblastoid fibroblasts and subcapsular cells into free hemocytoblastoid cells.