抗体库优化策略对特异性抗肝癌单链抗体的人源化

目的:对特异性抗肝癌单链抗体(single chain variable fragment,scFv)DM同步进行人源化和优化,以获得亲和力高和免疫原性低的人源化抗肝癌单链抗体.方法:通过分子结构和序列分析,确定对抗原结合位点的构象有重要影响的骨架区(FR)残基,把难以判断回复突变残基的人、鼠源副本同时编入人源化抗体序列中;通过重叠延伸PCR技术合成人源化抗体全基因,利用噬茵体展示技术构建人源化组合抗体库;通过肝癌细胞筛选阳性克隆,ELISA方法测定各个克隆抗原结合活性;用硫氰酸盐洗脱法测定并比较亲本抗体和人源化scFv的相对亲和力.结果:成功构建人源化组合抗体库,实际库容4.77×10 5,包含由2 5不同重链和2 2不同轻链组成的128种人源化DM;经过筛淘及ELISA鉴定,获得HDM1、HDM2和HDM3 3个阳性克隆,相对亲和力指数分别为HDM1 1.6 mol/L、HDM2 1.2 mol/L和HDM3 2.2 mol/L.结论:抗体库优化法是一个高效、简便的人源化方法;获得的3株阳性克隆HDM1、HDM2、HDM3与亲本抗体DM同样具有良好的抗原结合特异性和较高的亲和力.     

丙型肝炎病毒非结构蛋白NS5抗独特型人源单链可变区抗体的筛选与鉴定

目的：制备丙型肝炎病毒（HCV）非结构蛋白NSS（NS5）的抗独特型单链可变区抗体scFv(抗－Id scFv)，为研制HCV NS5的抗－Id scFv疫苗奠定基础。方法：采用噬菌体表面展示技术，将HCV NS5单克隆抗体固相包被于Nunc板，从噬菌体单链可变区抗体库中经过5轮“吸附－洗脱－扩增”筛选过程，随机挑选80个克隆，利用酶联免疫吸附试验（ELISA）、交叉反应和竞争抑制实验，对其进行免疫学检测，获得与HCV NS5单克隆抗体结合活性较强的抗－Id scFv阳性克隆，并对HCV NS5特异性抗－Id scFv的编码序列进行序列测定分析。结果：筛选得到的HCV NS5抗－Id scFv片段由786bp组成，具有结合HCV NS5单克隆抗体的生物学活性和特异性。结论：用噬菌体抗体库技术能够成功地获得HCV NS5的抗－Id scFv。本实验结果为开展用抗－Id scFv防治丙型肝炎的研究创造了条件。     

抗SSA噬菌体抗体库的构建及鉴定

目的　用噬菌体展示技术构建抗SSA单链抗体库。方法　分离抗SSA抗体阳性病人外周血单个核细胞 ,提取RNA并反转录cDNA。扩增免疫球蛋白重链可变区 (VH)和κ链可变区(Vκ)。通过重叠聚合酶链反应 (PCR)用连接片段将VH和Vκ体外连接成单链抗体 (single chainFv ,scFv)基因。而后用SfiⅠ和NotⅠ酶切并与噬菌粒 pHEN2 连接 ;将 pHEN2 scFv电转至大肠杆菌TG1,建立抗SSAscFvcDNA库。随机挑选克隆用PCR扩增插入片段了解插入效率。用辅助噬菌体VCS M13感染含 pHEN2 scFv的TG1,产生scFv噬菌体抗体。用酶联免疫吸附 (ELISA)检测构建的scFv噬菌体抗体库中有无抗SSA抗体存在。结果　与VH和Vκ支架结构互补的引物可扩增出 30 0～ 4 0 0bp大小VH和Vκ片断 ;重叠PCR体外连接成约 80 0bp大小scFv基因 (VH linker Vκ)。将scFv基因克隆至 pHEN2 ,电转TG1后计数克隆数为 3 0× 10 7cfu。随机挑选 12个克隆 ,其中 11个克隆PCR扩增出插入片段 ,插入效率约为 91%。用辅助噬菌体VCS M 13感染含 pHEN2 scFv的TG1后 ,产生 1 2× 10 14 pfu/ml噬菌体抗体颗粒。抗SSAELISA试剂盒检测其抗SSA活性 ,scFv噬菌体抗体的A值比相同稀释度的VCS M13的A值高 2 0～ 2 2倍。结论　建立的抗SSAscFv噬菌体抗体库含有的独立克隆数为 3 0× 10     

抗肝癌单链抗体体外亲和力成熟的改造及意义

目的从体外模拟抗体的体内成熟过程,以获得高亲和力抗肝癌单链抗体。方法采用错配聚合酶链反应(PCR)法随机突变抗肝癌单链抗体重链和轻链可变区,构建抗肝癌噬菌体抗体次级突变库,利用噬菌体展示技术从中筛选出高亲和力单链抗体突变株。结果抗肝癌噬菌体抗体次级突变库容为4．5× 10~7,经过3轮富集筛选和酶联免疫吸附法鉴定获得2株突变株M90、M116,不仅保留原始克隆的特异性,而且相对亲和力指数分别为原始克隆的1．7倍和2倍。结论错配PCR结合噬菌体展示技术是提高单链抗体亲和力的一种有效且简便的手段。     

血清甲状腺自身抗体、碘摄入量与Graves病发病及临床转归的关系

目的探讨血清甲状腺刺激性抗体（TSAb）、甲状腺刺激阻断性抗体（TSBAb）等甲状腺自身抗体、碘摄入量与Graves病（GD）甲状腺功能亢进症（甲亢）发病和转归的关系。方法测定3个不同碘摄入量地区63例临床甲亢患者的血清TSAb、TSBAb、促甲状腺激素结合抑制免疫球蛋白（TBⅡ）、甲状腺过氧化物酶抗体（TPOAb）和甲状腺球蛋白抗体（TGAb）,2年后随访。TSAb和TSBAb采用转染了重组人促甲状腺素受体的中国仓鼠卵巢细胞（rhTSHR—CHO细胞）生物法测定。结果初访GD甲亢患者TSAb、TBⅡ和TSBAb的阳性率分别为80．9％、61．7％和6．4％,TSAb和TBⅡ任-抗体阳性率为91．5％,显著高于对照组。TSAb和TBⅡ的一致率为59．6％。GD甲亢患者TSAb与TBⅡ（r=0．407）、甲状腺球蛋白（r=0、301）、甲状腺体积（r=0．317）正相关。初访碘过量地区GD甲亢患者TSAb阳性率（91．7％）显著高于轻度碘缺乏地区（66．7％）,3个地区GD患者TBⅡ、TPOAb、TGAb阳性率、活性和甲状腺体积无统计学差异。随访时患者分为甲状腺功能恢复组（G1）和未恢复组（G2）。G1组TSAb活性和甲状腺体积显著下降。当TPOAb滴度显著增高,且随访时继续保持高滴度时,甲状腺功能不易恢复。此时TSAb对甲状腺功能的影响降为次要因素。结论TSAb对GD甲亢的诊断和判断临床转归的意义大于TBⅡ,联合应用二者能提高GD甲亢患者促甲状腺激素受体抗体的检出率;GD甲亢的转归与TSAb、TPOAb浓度和甲状腺体积有关。     

甲状腺刺激抗体对甲状腺细胞分泌功能影响的信号途径

目的探讨甲状腺刺激抗体(TSAb)对甲状腺细胞分泌功能影响的信号途径。方法选择福建医科大学附属协和医院2003-01~10的Graves病(GD)40例,作为观察组;以院内健康职工40人,作为对照组。应用以酶联免疫吸附法(ELISA)测定蛋白激酶A(PKA)和蛋白激酶C(PKC)活性;应用PKA、PKC激活和抑制剂激活和阻断信号通路;应用放免法检测T3。结果(1)TSAb呈时间剂量依赖性激活甲状腺细胞中的PKA和PKC(P<0.05)。(2)TSAb刺激甲状腺细胞T3分泌的效应与PKA激活剂相似,且可被PKA抑制剂抑制,PKC激活剂佛波酯(TPA)和抑制剂无此效应。TPA可部分抑制TSAb和PKA激活剂刺激的T3分泌。结论TSAb刺激甲状腺细胞T3分泌主要是通过环腺苷酸(cAMP)-PKA途径,PKC途径可影响cAMP-PKA途径而抑制TSAb刺激的T3分泌。     

人源性老年痴呆单链噬菌体抗体库的构建

目的 构建人源性老年痴呆(AD)单链噬菌体抗体库,为进一步筛选AD相关抗原的人源性抗体奠定基础.方法 从200 ml AD病人的外周血中分离B淋巴细胞,提取总RNA,经逆转录合成总cDNA.以PCR技术,利用特定的引物分别扩增出人抗体的重链和轻链可变区基因片段(VH、VL),并分别克隆入噬菌粒pDAN5中,构建出含人单链抗体可变区(scFv)基因序列的pDAN5克隆载体,电转化感受态大肠杆菌XLI-Blue后,经辅助噬菌体M13K07超感染后回收全部重组噬菌体,构建成初级噬菌体抗体库.从抗体库中随机挑选数个克隆,提取质粒,PCR扩增目的片段后,用内切酶BstN Ⅰ消化,每个克隆经消化后的DNA指纹印迹用琼脂糖凝胶电泳分析,评价抗体库的多样性.结果 所有抗体VH和VL基因片段均得到了扩增并成功克隆及转化,经辅助噬菌体感染后,构建成初级抗体库.BstN Ⅰ消化后的DNA指纹图谱显示各克隆抗体基因各不相同,经计算构建的噬菌体抗体库容量为1.5×106.结论 利用噬菌体抗体库技术成功构建了AD病人的单链可变区噬菌体抗体库.     

人源性阿尔茨海默病噬菌体单链抗体库的构建

目的构建人源性阿尔茨海默病(AD)噬菌体单链抗体(scFv)库,为筛选β淀粉样蛋白(Aβ1-42)的人源性特异性抗体奠定基础。方法采集18例AD患者的外周血40ml,提取总RNA,应用RT-PCR法得到人抗体可变区重链(VH)和可变区轻链(VL)基因。将VH和VL由连接肽连接得到scFv片段,将所得片段双酶切后,克隆至pCANTAB5E噬菌体载体,大肠埃希菌TG1感受态细胞经电击转化,辅助噬菌体M13K07拯救后构建scFv型噬菌体抗体库。结果总RNA经逆转录PCR扩增VH和VL可变区基因的凝胶电泳显示,PCR产物长度分别为360bp和300bp,其连接形成的scFv片段长度为750bp,最终构建了库容为2.4×109的scFv库。BstNⅠ酶切鉴定构建的scFv库,经凝胶电泳可见,酶切片段长度差异性大,显示scFv库具有良好的多样性。结论成功构建人源性AD噬菌体scFv库,为进一步筛选Aβ特异性抗体,继而为AD的治疗研究奠定基础。     

促甲状腺素受体抗体检测的临床意义

促甲状腺素受体抗体(TRAb)与多种自身免疫性甲状腺疾病的发病密切相关,是临床诊断这些疾病的重要指标.TRAb在功能上可分为甲状腺刺激性抗体(TSAb)和甲状腺刺激阻断性抗体(TSBAb),可与促甲状腺素受体分子上不同位点结合产生不同生物学效应.其中TSAb可引起Graves病(GD)患者甲状腺功能亢进和弥漫性甲状腺肿的发生,而TSBAb则可导致自身免疫性甲状腺功能减退症(甲减)患者甲减的发生.本文将对TRAb检测在GD鉴别诊断、辅助制定GD治疗方案、抗甲状腺药物疗效及预测复发、筛查自身免疫性甲减等方面的临床价值作一全面综述.     

甲状腺刺激抗体与Graves眼病临床特点的关系

比较分析32例初诊的Graves眼病（GO）患者和27例无眼病Graves病（GD）患者甲状腺刺激抗体（TSAb）与临床特点的关系；并且分析影响糖皮质激素冲击治疗GO预后的因素，发现GO组TSAb水平明显高于GD组，且对预后有显著影响，提示TSAb可能是TSH受体抗体中与眼病损伤更为密切的抗体成分，可作为GO的预测指标。     

Graves' disease following hypothyroidism due to Hashimoto's disease: studies of eight cases

Hashimoto's and Graves' diseases represent the main two types of autoimmune thyroid disease. The combination of these two is well known. However, occurrence of Graves' disease after primary hypothyroidism is rare. We report seven patients with hypothyroidism due to Hashimoto's disease, who developed Graves' disease with hyperthyroidism. We also report one patient with hypothyroidism due to Hashimoto's disease, who continued to be hypothyroid even in the presence of TSAb (thyroid stimulating antibody). These patients were divided into three groups according to the changes in thyroid function and clinical course: (1) transient hyperthyroidism due to Graves' disease following hypothyroidism; (2) persistent hyperthyroidism due to Graves' disease following hypothyroidism; and (3) persistent hypothyroidism with positive TSAb. Such changes in thyroid function and clinical course seem to be decided by three factors: (1) TSAb and (2) TSBAb (thyroid stimulation blocking antibody) activities in the blood and (3) the responsiveness of the thyroid gland to TSAb. Seven patients had hyperthyroidism, when they had TSAb, which stimulated the thyroid gland; one of these seven patients had TSBAb during the hypothyroid state and TSAb during the hyperthyroid state, indicating that the alterations in the thyroid state related to the balance between the activities of TSAB and TSBAb. Another patient continued to be hypothyroid despite the presence of TSAb; his thyroid gland was not palpable and could not respond to TSAb.     

Thyroid-stimulating immunoglobulins. The related antigens

The presence of thyroid stimulating antibodies (TSAb) in patients with Graves' disease is well established. Considerable evidence has accumulated that these are antibodies to thyroid plasma membrane components related to the TSH receptor. The question of whether thyroid stimulation is mediated by a direct interaction with the TSH binding site is still debated. Recent data obtained by the use of monoclonal antibodies to the TSH receptor are consistent with the view that Graves' immunoglobulins comprise antibodies to at least two different components of the TSH receptor complex, one of which is more strictly related to the binding of TSH and the other to the transmission of the hormonal effect. The causative role of TSAb in the hyperthyroidism of Graves' disease is widely recognized. The use of human specific stimulation assays has circumvented the objection of the relatively low frequency of LATS-positive patients. Individual variations in the thyroid response may account for the lack of correlation between levels of thyroid stimulating immunoglobulins and most parameters of thyroid function. In this respect, the interference of non-stimulatory thyroid antibody and of other autoimmune mechanisms may be of importance. An important clinical implication of TSAb and TSH-binding inhibiting antibody determinations is their prognostic value in predicting the relapse of hyperthyroidism in treated patients. This clinical application has been so far limited by the technical difficulty of the assays. This emphasizes the need for a simple and reliable test, which can be used for routine measurements of TSAb.     

STUDIES ON THYROTROPHIN RECEPTOR ANTIBODIES IN PATIENTS WITH EUTHYROID GRAVES'' DISEASE

Thyroid stimulating antibodies (TSAb) and TSH-binding inhibitor immunoglobulins (TBII) were assessed in 30 patients with euthyroid Graves' disease. TSAb were detected in 24 cases (80.0%), the incidence being not significantly different from that in hyperthyroid Graves' disease (29/30, 97.6%). On the other hand, the incidence of TBII in patients with euthyroid Graves' disease (12/30, 40.0%) was significantly lower than that in patients with hyperthyroid Graves' disease (30/30, 100.0%). The mean TSAb and TBII activities in the euthyroid patients were significantly lower than in the hyperthyroid patients (P less than 0.005 and P less than 0.001, respectively). Both TBII and, more closely, TSAb activities correlated with T3-nonsuppressibility and inhibition of serum TSH response to TRH stimulation. The findings supported the stimulation in vivo of the thyroid by these antibodies. Both antithyroglobulin and antimicrosomal antibody titres in euthyroid Graves' disease were significantly lower than in hyperthyroid Graves' disease (P less than 0.05, P less than 0.01, respectively). Goitre size was significantly smaller (P less than 0.001), and 99mTc thyroid uptake was significantly lower (P less than 0.001) in the euthyroid than in the hyperthyroid group. Thus, the reduced mass of thyroid tissues responding to the stimulators was considered to be one of the factors responsible for the euthyroidism despite the presence of TSAb. The high incidence of TSAb and relatively low incidence of TBII in euthyroid Graves' disease indicate that the presence of TSAb does not necessarily lead to hyperthyroidism and that the development of overt thyrotoxicosis may require augmentation of both TSAb and TBII.     

Interactions between TSH binding inhibiting--and adenylate cyclase stimulating--antibodies in Graves' disease

It is well known that there exists a dissociation between TSH binding inhibiting antibody (TBIAb) and thyroid stimulating antibody (TSAb) in some patients with Graves' disease. The present studies were undertaken to investigate the interaction between TBIAb and TSAb quantitatively using porcine thyroid cells in order to determine whether the binding sites of TBIAb and TSAb are identical or independent from each other. TBIAb was determined using solubilized porcine thyroid membrane and TSAb using cultured porcine thyroid cells, and both were expressed as equivalent amounts of bovine TSH. In order to avoid interassay variations, all determinations were performed with a single batch of porcine thyroids. Serum samples were obtained from ten untreated patients with Graves' disease; two were negative for TBIAb and TSAb, two were TBIAb negative and TSAb positive, two were weak positive for both, two were strong positive for both, and two were TBIAb strong and TSAb weak positive. IgG from each patient were mixed with equal amounts of IgG of the other nine patients. In order to observe interactions, measured values for TBIAb and TSAb of mixed samples were compared to estimated values (addition of values of original samples). The correlation coefficient between measured values and estimated values for TBIAb was +0.964, and that for TSAb was +0.989. These results that TBIAb does not interfere with the activity of TSAb and vice versa suggest that at least in some patients with Graves' disease, TBIAb and TSAb are different antibodies which have different binding sites.     

Immunoglobulins of untreated Graves' patients with or without thyrotropin receptor antibody (determined by porcine thyrocytes) universally elicit potent thyroid hormone-releasing activity in cultured human thyroid follicles.

Thyrotropin receptor antibody (TRAb), comprising thyrotropin binding inhibitor immunoglobulin (TBII) and thyroid-stimulating antibody (TSAb), both of which are conventionally determined using porcine thyrocytes in Japan, is not always positive in patients with untreated Graves' disease. To elucidate whether immunoglobulin G (IgG) obtained from TBII/TSAb-positive (+) or negative (-) Graves' disease patients are responsible for hyperthyroidism, we investigated the thyroid hormone-releasing activity (THRA) of these IgGs in human thyroid follicles in suspension culture, in which bovine thyrotropin (bTSH) is detectable even at 0.1 microU/mL. Human thyroid follicles, obtained from Graves' disease patients by subtotal thyroidectomy, were cultured in serum-free F-12/RPMI-1640 medium supplemented with bTSH or purified Graves' IgGs. After preculturing for 3 days, 125I was added, and after an additional 3 days of culture, 1251 incorporated into the thyroid follicles and organic 125I released into the culture medium (mainly 1251 -T4 + 125I-T3) were counted. Seventy TBII(+)/TSAb( + )-, 3 TBII( + )/TSAb( - )-, and 3 TBII( - )/TSAb( + )- patients with untreated Graves' disease were all positive for THRA, which became undetectable in spontaneous remission obtained after several years of medical treatment. The THRA was equivalent to 0.8-230 microU/mL bTSH. Furthermore, 2 TBII(-)/TSAb(-) patients were significantly positive for THRA. This TBII(-)/TSAb(-)IgG stimulated human thyrocytes to produce cyclic adenosine monophosphate (cAMP), and this was partially inhibited by antihuman IgG antibody. The THRA induced by TBII(+)/TSAb(+) IgGs as well as TBII(-)/TSAb(-) IgG was inhibited by blocking-type TRAb obtained from TBII(+) patients with myxedema. There was a significant correlation between THRA and TSAb. These in vitro findings suggest that all IgGs obtained from untreated Graves' patients (n = 78) elicit potent THRA in human thyroid follicles in suspension culture. Because the TBII(-)/TSAb(-) IgGs can stimulate cAMP production in human but not in porcine thyrocytes, they probably recognize epitope(s) of TSH-binding sites specific to the human thyrotropin (hTSH) receptor. Furthermore, we have demonstrated that the thyroid gland of hyperthyroid Graves' patients is stimulated by IgG(s) equivalent to at least 0.8 microU/mL bTSH (about 5 microU/mL hTSH) in vitro.     

Treatment with triiodothyronine (T3) against multinodular goiter fails to prevent the onset of Graves' disease

We report two cases who developed hyperthyroidism and positive thyroid stimulating antibody (TSAb) while they were treated with T[3] under the diagnosis of follicular adenoma of thyroid for four or eleven years, respectively. In both cases thyroid scintigraphy revealed an increased radioiodine uptake with multinodular defects. The uptake of 131I was not suppressed by the administration of T[3]. Histological examination showed encapsulated nodule surrounded by diffuse hyperplasia. TBII or TSAb appeared in accordance with increased plasma levels of T[3] and T[4]. In spite of suppressed TSH the appearance of TSAb and subsequent manifestations of Graves' disease suggested that T[3] failed to prevent the immune mechanism which was involved in the production of TSAb.     

Fetal and neonatal hyperthyroidism and hypothyroidism due to maternal TSH receptor antibodies.

Autoimmune thyroid disease is a generic term that includes Graves' disease and Hashimoto's thyroiditis. In the former, there is overactivity of the thyroid due to the action of a thyroid-stimulating antibody (TSAb). Pathogenesis of Hashimoto's thyroiditis is largely cell-mediated immune destruction of the thyroid. Nonetheless, there may be either a goiter or an atrophic gland. There is evidence that in some patients the lack of goiter is associated with the presence in the blood of an antibody that inhibits the binding of TSH to its receptor. This TSH-binding inhibiting antibody (TBIAb), therefore, prevents TSH from stimulating the thyroid and constitutes an acceptable explanation for an agoitrous state. Collectively, TSAb and TBIAb, both of which are IgG, are known as TSH receptor antibodies (TRAb).     

老年甲亢患者甲状腺刺激抗体检测及临床应用

以血清ＩｇＧ刺激培养的甲状腺机能亢进症患者甲状腺细胞，通过测定ｃＡＭＰ的释放值确定甲状腺刺激抗体（ＴＳＡｂ）的活性。结果显示：①老年Ｃｒａｖｅｓ病（ＧＤ）未治组ＴＳＡｂ活性显著高于ＧＤ缓解组与缓解停药组，３组患者ＴＳＡｂ的阳性率分别为８８．９％、４７．８％和４５．５％，甲状腺腺瘤组和对照组ＴＳＡｂ均为阴性。②ＧＤ缓解停药时５例ＴＳＡｂ阳性者中４例（８０．０％）于１年内复发，而６例阴性者仅１例（１６．７％）复发。③ＴＳＡｂ活性与血清Ｔ３、Ｔ４浓度无明显相关关系。提示ＴＳＡｂ测定对老年ＧＤ的诊断、鉴别诊断、治疗和预后判断有重要的指导意义。     

EVALUATION OF TSH RECEPTOR ANTIBODY BY ''NATURAL IN VIVO HUMAN ASSAY'' IN NEONATES BORN TO MOTHERS WITH GRAVES'' DISEASE

Neonatal thyrotoxicosis induced by transferred TSH receptor antibody (TRAb) is the ideal human in-vivo experimental system for the evaluation of TRAb. The clinical significance of circulating TRAb in Graves' disease was evaluated by this 'natural in-vivo human assay'. TRAb activity in vitro was measured by radioreceptor assay (thyrotrophin-binding inhibitor immunoglobulin, TBII) and sensitive cAMP accumulation assay using FRTL-5 cells (thyroid-stimulating antibody, TSAb). Further, the binding-stimulation index (B-S index) was newly introduced, which was the most useful indicator for prediction of neonatal thyrotoxicosis, calculated as the product of TBII and TSAb (Tamaki et al., 1988a). Maternal serum TRAb indices showed highly significant correlations with the serum free T4 index (FT4I) and free T3 index (FT3I) in neonates (5-10 days after birth) born to 20 mothers with Graves' disease who had positive TBII and/or TSAb (FT4I: r = 0.825 for TBII, r = 0.908 for TSAb, r = 0.944 for the B-S index, P less than 0.001; FT3I: r = 0.622 for TBII, P less than 0.01, r = 0.812 for TSAb, r = 0.791 for the B-S index, P less than 0.001; n = 20). In contrast, in 57 untreated adult patients with hyperthyroid Graves' disease, the FT4I and FT3I levels were not correlated with any of the TRAb indices. The linear regression relationship between the B-S index and FT4I found in neonates was applied to values in adult patients with Graves' disease, and the patients were divided into three groups on the basis of the 95% confidence limit: high, normal, and low responders of thyroid hormone (FT4I) secretion to the B-S index. FT4I and the ratio of FT4I to the B-S index were highest and the TRAb indices were lowest in the high responders, while FT4I and the FT4I/B-S index ratio were lowest and the TRAb indices were highest in the low responders. The FT4I/B-S index ratio was inversely correlated with the titres of antithyroid microsomal antibody in all the adult patients with untreated Graves' disease (r = -0.288, P less than 0.05). The results suggest that in-vitro assays using animal thyroid cells and cAMP as an index of response are suitable for detecting circulating thyroid stimulating activity in vivo. Secretion of thyroid hormones in Graves' disease may be regulated not only by circulating thyroid-stimulating antibodies but also by intrathyroidal stimulatory factors or by inhibitory or destructive factors.