Michelangeli V P, Munro D S, Poon C W, Frauman A G, Colman P G
Department of Diabetes and Endocrinology, Royal Melbourne Hospital, Parkville, Australia.
Clin Endocrinol (Oxf). 1994 May;40(5):645-52. doi: 10.1111/j.1365-2265.1994.tb03017.x.
Most bioassays used to measure thyroid stimulating antibodies (TSAb) rely on the ability of patient sera to generate cAMP in cultured cells of non-human origin. The cell line most commonly used has been the rat thyroid cell line, FRTL-5. Recently, a new cell line (JPO9) possessing a transfected human TSH receptor has been developed. The aim of this study was to evaluate the JPO9 cells by comparing them with the widely used FRTL-5 cells, using the method of 3H-adenine incorporation for the direct measurement of intracellular cAMP.
The ability of patient sera to generate cAMP production in JPO9 and FRTL-5 cells was used as the index of thyroid stimulation. The specificity of the assay was determined using sera from patients with autoimmune thyroid and non-thyroid autoimmune diseases.
We studied sera from 28 patients with newly diagnosed Graves' disease, 72 patients with Graves' disease at various stages of treatment, 8 patients with Hashimoto's thyroiditis, 40 patients with a variety of non-thyroid autoimmune disease and 42 control subjects.
The intracellular 3H-cAMP generated in JPO9 cells following preincubation with 3H-adenine and subsequent incubation with patient or control sera, was used as a measure for TSAb. The results obtained with these cells were compared to those obtained with the widely used FRTL-5 cells and they were also correlated with the measurement of TSH binding inhibitory immunoglobulins by a conventional radioreceptor assay.
JPO9 cells were more sensitive and had a wider range of response to bovine TSH (bTSH) than had FRTL-5 cells (10(-7)-10(-2) U/ml compared to 10(-5)-10(-3) U/ml respectively). Unlike FRTL-5 cells, JPO9 cells respond well to unmodified serum and grow constitutively in the absence of TSH, thereby not requiring TSH deprivation prior to assay. The TSAb values obtained with both cell lines correlated well (r = 0.87). The JPO9 cells responded specifically to Graves' sera (23 out of 28 newly diagnosed patients were positive), whereas no patients with Hashimoto's thyroiditis and only 4 of 40 patients with non-thyroid autoimmune diseases gave low positive results for the presence of TSAb.
The JPO9 cells provide similar diagnostic information to FRTL-5 cells in patients with autoimmune thyroid disease. However, because they are more sensitive, grow faster, have less fastidious growth requirements and respond to unextracted sera, compared to FRTL-5 cells, we conclude that the JPO9 cells are preferable for the measurement of TSAb.
大多数用于检测促甲状腺素抗体（TSAb）的生物测定法依赖于患者血清在非人类来源培养细胞中产生环磷酸腺苷（cAMP）的能力。最常用的细胞系是大鼠甲状腺细胞系FRTL-5。最近，一种转染了人促甲状腺激素（TSH）受体的新细胞系（JPO9）已被开发出来。本研究的目的是通过将JPO9细胞与广泛使用的FRTL-5细胞进行比较，采用³H-腺嘌呤掺入法直接测量细胞内cAMP，来评估JPO9细胞。
将患者血清在JPO9和FRTL-5细胞中产生cAMP的能力用作甲状腺刺激指标。使用自身免疫性甲状腺疾病和非甲状腺自身免疫性疾病患者的血清来确定该测定法的特异性。
我们研究了28例新诊断的格雷夫斯病患者、72例处于不同治疗阶段的格雷夫斯病患者、8例桥本甲状腺炎患者、40例各种非甲状腺自身免疫性疾病患者的血清以及42名对照者的血清。
在³H-腺嘌呤预孵育后，再与患者或对照血清孵育，然后测量JPO9细胞内产生的³H-cAMP，以此作为TSAb的测量指标。将这些细胞获得的结果与广泛使用的FRTL-5细胞获得的结果进行比较，并且还将它们与通过传统放射受体测定法测量的TSH结合抑制性免疫球蛋白进行相关性分析。
与FRTL-5细胞相比，JPO9细胞对牛TSH（bTSH）更敏感，反应范围更广（分别为10⁻⁷ - 10⁻² U/ml和10⁻⁵ - 10⁻³ U/ml）。与FRTL-5细胞不同，JPO9细胞对未处理的血清反应良好，并且在没有TSH的情况下能持续生长，因此在测定前无需剥夺TSH。两种细胞系获得的TSAb值相关性良好（r = 0.87）。JPO9细胞对格雷夫斯病血清有特异性反应（28例新诊断患者中有23例呈阳性），而桥本甲状腺炎患者无一例呈阳性，40例非甲状腺自身免疫性疾病患者中只有4例TSAb呈低阳性结果。
在自身免疫性甲状腺疾病患者中，JPO9细胞提供了与FRTL-5细胞相似的诊断信息。然而，与FRTL-5细胞相比，由于它们更敏感、生长更快、生长要求不那么苛刻且对未提取的血清有反应，我们得出结论，JPO9细胞更适合用于TSAb的测量。
