Characterization of thyroid-stimulating immunoglobulin-induced cyclic AMP accumulation in the rat thyroid cell strain FRTL-5: potentiation by forskolin and calibration against reference preparations of thyrotrophin

Abstract

A clonal strain of rat thyroid cells (FRTL-5) has been used to investigate the biological activity of a Research Standard preparation of long-acting thyroid stimulator (LATS-B). Using the accumulation of intracellular cyclic AMP as a response parameter, significant stimulation was attained at a LATS-B dose of 0.75 mu./ml. The inter-bioassay coefficient of variation in response to a fixed dose of LATS-B (1.25 mu./ml) was 20.5%, as determined using eight sequential subcultures. Cells cultured directly from frozen stocks responded to both bovine TSH and LATS-B in a manner indistinguishable from cells subjected to regular subculturing. Cyclic AMP responses to incremental doses of LATS-B were potentiated after the inclusion of a low dose of forskolin (0.1 mumol/l). However, forskolin addition had no effect on the time-course of LATS-B-stimulated cyclic AMP accumulation, half-maximal responses being attained after 60 min in either the presence or absence of the diterpene. In the presence of 0.1 mumol forskolin/l, intracellular cyclic AMP responses to LATS-B were demonstrably parallel with those to human TSH (Second International Reference Preparation, 80/558), whilst parallel incremental cyclic AMP responses were also observed in respect of TSH and serial dilutions of a potent thyroid-stimulating immunoglobulin (TSIg) preparation, indicating that for this particular Graves' disease patient, TSIg bioactivity may be expressed in terms of a convenient and reproducible standard, as TSH microunit equivalents.

A sensitive and practical assay for thyroid-stimulating antibodies using FRTL-5 thyroid cells

“…In the present study, we developed and evaluated a new TSAb assay using FRTU-5 cells in hypotonie medium as responders and crude Ig fractions prepared by PEG precipitation as stimulators. The reproducibility of the assay was satisfactory for a bioassay, the coefficient of variation obtained being similar to that reported by Bidey et al (1985).…”

“…In comparison with an assay for TSAb using thyroid cells in primary culture, the assay using this continuously proliferating thyroid cell strain (Vitti et al 1983;Bidey et al 1985), seems more promising as a routine clinical test, the cells showing more stable and reproducible cAMP responses to the thyroid stimulators. In the present study, we developed and evaluated a new TSAb assay using FRTU-5 cells in hypotonie medium as responders and crude Ig fractions prepared by PEG precipitation as stimulators.…”

Balb/C mice immunized with either xenogeneic or syngeneic TSH produce immunoglobulins with anti-TSH and thyroid-stimulating activities

“…nmol/1 forskolin (Calbiochem-Behring, La Jolla, CA)(15), and 5 pmol/1 isobutyl-methyl-xanthine (MIX) (Sigma, St. Louis, MO). For calibration, bTSH (Sigma) was used in the range of 0.25-2 pU/well.…”

Anticorpos anti-tiróide: aspectos metodológicos e importância diagnóstica

“…Quanto à baixa concentração dos anticorpos, esta é uma limitação que deve ser vencida com a utilização de ensaios cada vez mais sensíveis, desde que a concentração de anticorpos anti-TR se situa na faixa de ng/mL, enquanto as de anticorpos anti-TPO são, em geral, de 100 a 1000 vezes superiores (53). Do ponto de vista técnico, as metodologias empregadas podem ser divididas em três tipos principais: métodos baseados em inibição da ligação de TSH marcado a receptor isolado, em fase líqüida ou sólida, ou em células que expressam o receptor naturalmente ou por transfecção (52,(54)(55)(56); métodos baseados na medida do efeito biológi-co observado em células expressando receptor de TSH naturalmente ou por transfecção (17,(57)(58)(59); métodos baseados em citometria de fluxo (60). Cada uma destas variantes metodológicas tem vantagens e desvantagens, e aplicações específicas.…”