Standardization of serum free media for cryopreservation of RK13 cells.

Foetal calf serum present in the media used for cryopreservation was replaced by various synthetic polymer such as gelatin, glycerol, carboxymethyl cellulose and dimethyl sulphoxide at various concentration. Growth pattern of cells, % survival and karyological studies have been done in the present study. It was found that optimum concentration of carboxymethyl cellulose was 0.1% in combination with 10% glycerol and 10% DMSO. At this concentration percentage survival of cells was found maximum and karyotype was found normal without any abnormality in the chromosomes. It was concluded from the study that serum free media can be employed for the cryopreservation of these cells which are further used for production of tissue culture vaccines without causing any adverse affects.

Development of cultivation technique for pure Plasmodium falciparum gametocytes.

Pure gametocyte culture of Plasmodium falciparum, isolate KT3, from Kanchanaburi Province, Thailand, was successfully established by 5% sorbitol treatment on days 9, 10 and 11 following initiation of culture. Using medium supplemented with 15% human plasma and activated erythrocytes, daily medium change was not required during the cultivation. There was 99% reduction in the numbers of asexual parasites in the culture but the numbers of gametocytes were not affected. Furthermore, the gametocytes could undergo their usual morphological development with retention of function as demonstrated by the appearance of exflagellating microgametocytes, macrogametocytes and of oocyst formation in midgut of infected mosquito.

Anti-dengue IgG detection by an indirect ELISA.

Protein-free culture media were originally developed for hybridomas to simplify downstream processing and purification. For the same reasons, we have used these protein-free media for passaging dengue 2 virus in C6/36 cells. This provided us with an infected supernatant (DenPF) which could then be used as coating antigens for an indirect enzyme-linked immunosorbent assay (ELISA) to determine dengue IgG levels. Using this preparation, the main immunogenic band as seen by immunoblot appeared to be viral envelope protein (E). Without the high concentrations of "competing" proteins from fetal calf serum (FCS), the Den2PF could be directly coated onto 96-well ELISA plates. The amount of viral proteins in Den2PF appeared to be sufficient so that there was no need for further purification steps, eg polyethylene glycol (PEG) precipitation, which made this preparation cost effective. It compared favorably with the dengue dot enzyme immunoassay (DEIA; sensitivity of 95.7% and specificity of 95.2%).