Taku Kashiwai

Immunological and biological characteristics of recombinant human thyrotropin

Analyses of epitopes and biological activity were made on two preparations of recombinant human thyrotropin produced in Chinese hamster ovary cells. Seven monoclonal antibodies recognizing four epitopes on alpha subunit of hTSH (hTSH alpha) and three on beta subunit (hTSH beta) were used for the analysis. Binding activities of the two rhTSH with each antibody were almost identical with that of a pituitary-derived reference hTSH, except at one epitope on hTSH alpha. Their immunoreactivity measured by four commercial immunoassay kits and their bioactivity by thyrotropin dependent FRTL-5 cell system, however, agreed closely with those of the reference hTSH. From these results and its constant availability, rhTSH will be a good candidate for a future standard material in assays for hTSH.

The stability of immunological and biological activity of human thyrotropin in buffer: its temperature-dependent dissociation into subunits during freezing.

The stability of thyroid-stimulating hormone (TSH) in buffer under various storage conditions was studied with regard to its immunoreactivity measured by an immunoradiometric assay and its bioactivity by a sensitive FRTL-5 cell bioassay. The immunoreactivity was well retained at 4 degrees C or 24 degrees C throughout the study period of 90 days. At -20 degrees C, however, it decreased proportionately with the storage time. The mean reduction was 42.1% at 90 days compared with that when stored at -80 degrees C. The bioactivity showed a similar course of change with its reduction of 44.3% at -20 degrees C in 90 days. The loss of both activities was attributed to the dissociation of human (h) TSH molecule into its subunits. The concentration of the alpha subunit of hTSH in those samples stored at -20 degrees C gradually increased from the initial undetectable level to that almost equivalent on a molar basis to the loss of immunoreactivity. The enrichment of albumin in the buffer to a level of more than 1.0% was effective in preventing the occurrence of such a phenomenon. These data indicate that hTSH, when frozen at -20 degrees C in buffer is gradually dissociated into its subunits, despite its outstanding stability for 90 days at both 4 degrees C and 24 degrees C. However, no apparent inconsistency between immunological and biological activities, was observed at any temperature between -20 degrees C and 24 degrees C.