A rapid and sensitive method for the detection of mycoplasmas in infected cell cultures using 6-methyl purine deoxyriboside.

6MPDR causes the death of cells lightly infected with mycoplasmas. This is brought about by the action of the mycoplasmal enzyme adenosine phosphorylase which converts 6MPDR to highly toxic metabolites. If the medium conditions are adjusted to favour the growth of mycoplasmas by the addition of pig serum to the medium, infections as low as 1 mycoplasma per 200,000 cells can be detected within 7 days. This finding enables mycoplasma tests to be carried out rapidly and reliably by untrained personnel.

Determination of the activity of clinical grade human growth hormone preparations by in vivo bioassay, in vitro cell proliferation assay and solid phase radioimmunoassay.

Preparations of clinical grade human growth hormone (hGH) have been examined for activity by an in vitro bioassay using NB2 lymphoma cells and by a monoclonal antibody (NA 71)-based radioimmunoassay. The activities observed have been compared with those obtained by the in vivo growth bioassay performed in mice of the Snell dwarf strain. The two in vitro assays correlated well with each other for specific preparations of hGH, although non-parallelism was observed between different preparations of the hormone. Some preparations of hGH were highly potent in the NA 71 immunoassay, but not in the NB2, cell-proliferation assay, suggesting the presence of antigenically active but biologically inactive hormone. Chromatographic studies on early hGH preparations revealed the presence of dimetric, trimetric and aggregated hormone as well as small quantities of prolactin. These were studied individually and shown to diverge in potency in the respective assays. It is concluded that the correlations between the in vivo bioassay in dwarf mice and the in vitro assays may be compromised by the varying potency of the constituent forms of the hormone and the non-linearity of the in vivo growth assay.

The titration of clostridial toxoids and antisera in cell culture.

The practicability of cell culture titration methods for antigens and antitoxins derived from Clostridium perfringens, Cl. septicum and Cl. novyi was investigated. Toxin neutralization could not be demonstrated with Cl. perfringens filtrates but assays for antitoxin based on the use of cultures of Vero cells were practicable for Cl. novyi and Cl. septicum. In the case of the latter, the precision and reproducibility of the test was sufficient for quality control purposes.

The characterization of three monkey kidney cell lines.

Three monkey kidney cell lines, Vero, GL-V3 and MA-104 were subjected to karyological analysis to determine their chromosomal stability and to confirm their species of origin. Although the lines were shown to be relatively stable throughout all of the passage levels that were tested, the species of origin of one of them was found to be different from that claimed by the originators. This finding was supported by data from isoenzyme studies.

Effect of bacterial toxins in serum on the chromosomes of WI-38.

Two batches of foetal calf serum, free from detectable bacterial, mycotic, mycoplasmal and bovine viral conta mination and possessing good growth-promoting properties induced an abnormally high number of chromatid breaks in WI-38 cells to the extent that the cells were unacceptable as a vaccine substrate. This phenomenon, immediately reversible on changing to a different batch of serum, was unaffected by pasteurisation, suggesting that it was caused by some unidentified toxic factor(s). It was found that the same effect could be brought about by the addition of subcytotoxic concentrations of some bacterial toxins to the culture media. These findings re-emphasise the importance of improved methods for the collection and the maintenance of sterility throughout the processing of such sera, especially when they are intended to be incorporated in media used to establish vaccine substrates.

The preparation and testing of antihuman lymphoblast globulin for clinical use.

Antibodies to cultured human lymphoblasts were raised in horses using a schedule employing both subcutaneous and intravenous routes of injection. Plasma from groups of horses was pooled and the IgG prepared from each pool was tested extensively for safety and immunosuppressive efficacy in vitro and in vivo. On the basis of the results of skin grafting in monkeys, only globulins derived from the first main bleeds were blended to produce a bulk for clinical use. One early pool of globulin was discarded because when undiluted, it was lethal in monkeys by the intravenous route, and another pool was discarded because it contained antibodies which bound to the glomerular basement membrane of rats. No signs of toxicity as judged by haematology, blood biochemistry, and histology were detected in monkeys receiving the clinical blend of globulin either subcutaneously or intravenously.