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Hazel Humphries
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Susan M. Dirmikis
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D. S. Munro
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A detailed comparison between the use of human and porcine thyroid membranes for the radioreceptor assay (RRA) of bovine TSH (bTSH) and thyrotrophin-binding inhibiting immunoglobulins (TBIIg) is reported. Bovine thyroid membranes were also investigated but were found to be far less satisfactory than either human or porcine thyroid membranes. The affinity constant (K a) of the interaction of bTSH with porcine thyroid membranes (K a = 3·3 × 109l/mol) measured by Scatchard analysis was higher than with human thyroid membranes (K a = 2·1 × 108l/mol). Porcine thyroid membranes were more sensitive for the assay of bTSH (detection limit 30 μu., half-maximal inhibition 0·3 mu.) than human thyroid membranes (detection limit 200 μu., half-maximal inhibition 7·4 mu.). Preincubation of membranes from either species with immunoglobulin rich in long-acting thyroid stimulator (LATS) inhibited the saturable binding of 125I-labelled TSH to a greater extent than did normal immunoglobulin. The binding of 125I-labelled TSH to porcine membranes was more sensitive to the inhibitory effect of LATS-immunoglobulin and was also less affected by normal immunoglobulin than was binding to human thyroid membranes. When assayed with each type of membrane preparation there was good correlation between the RRA of immunoglobulins prepared from patients with Graves's disease and from normal subjects (n = 18) (r = 0·85, P <0·001, n = 73). The incidence of positive TBIIg in untreated Graves's disease was greater for porcine than for human thyroid membranes.

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S. D. HOLMES
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S. M. DIRMIKIS
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T. J. MARTIN
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D. S. MUNRO
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Thyroid-stimulating immunoglobulins were prepared from two potent sera, one contained long-acting thyroid stimulator (LATS) and the other contained both LATS and LATS-protector (LATS-P). The potencies of the immunoglobulin G (IgG) preparations were estimated in the McKenzie assay.

The accumulation of cyclic AMP in mouse thyroid lobes was stimulated only by LATS–IgG; LATS-P–IgG was inactive. In contrast, both LATS–IgG and LATS-P–IgG were equally effective in slices of human thyroid.

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G. D. BROADHEAD
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S. M. DIRMIKIS
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H. HUMPHRIES
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S. K. JUSTICE
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G. LOY
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T. SMITH
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Section of Pharmacology, Academic Division of Medicine and *Field Laboratories, The University, Sheffield, S102TN

(Received 8 July 1975)

The McKenzie (1958) bioassay remains the principal tool for studying the long-acting thyroid stimulator (LATS) and LATS-protector (LATSP), although radioreceptor binding assays are being developed (Manley, Bourke & Hawker, 1974; Smith & Hall, 1974). A dog biscuit met the low-iodine requirements until 1974, when batches contained sufficient iodine to make them unsuitable for use in this bioassay. Bread contains negligible iodine, unless iodated dough conditioner is added (London & Vought, 1965). Thus an investigation was made of the suitability of bread for use in the McKenzie bioassay.

Female, white Swiss mice were bred as described by Loy & Broadhead (1968), the colony being fed on pasteurized breeding diet (Oxoid Ltd). Mice were weaned at 4 weeks, fed on diet 86 (Oxoid Ltd) until body weights were 15–20 g (i.e. usually for 1–2 weeks),

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S. D. HOLMES
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SUSAN M. DIRMIKIS
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T. J. MARTIN
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D. S. MUNRO
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The activation of adenylate cyclase and the accumulation of cyclic AMP resulting from the action of human thyroid-stimulating hormone (TSH), long-acting thyroid stimulator (LATS) or LATS-protector (LATS-P) have been investigated in preparations of human thyroid membranes and slices.

Human TSH significantly increased adenylate cyclase activity in membranes from non-toxic goitres whereas LATS and LATS-P had no consistent effect. However, pre-incubation of goitrous membranes with LATS–immunoglobulin G inhibited the effect of TSH on adenylate cyclase. When thyroid membranes were prepared from the glands of patients with Graves's disease neither TSH nor thyroid-stimulating immunoglobulins (TSIg) stimulated adenylate cyclase significantly.

Whether from non-toxic goitres or thyrotoxic tissue, the concentration of TSH needed to induce half of the maximum response was lower in thyroid slices than in membranes. Both LATS and LATS-P significantly stimulated the accumulation of cyclic AMP in slices of goitrous tissue but thyrotoxic tissue slices did not respond.

In goitrous slices, submaximum concentrations of TSH and TSIg caused additive responses in the accumulation of cyclic AMP but TSIg did not increase the maximum response to TSH.

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