以重组TrxTSHRc、TrxTSHRn蛋白为抗原ELISA检测人血清TRAb含量
摘要
自身免疫性甲状腺疾病(autoimmune thyroid disease,AITD)是一组发病机制复杂的器官特异性自身免疫疾病。人促甲状腺激素受体(hTSHR)是AITD的主要靶抗原,在AITD的发病中起重要作用。hTSHR基因定位于14号染色体(14q31)上,是一种G蛋白偶联受体,为大分子糖蛋白其肽链可分为膜外区,跨膜区和膜内区。TSHR膜外区(TSHR-ecd)为促甲状腺激素(TSH)或TSHR抗体(TRAb)识别及结合部位,有广泛的、多个不连续的结合位点。其中TSHR刺激型抗体(TSAb)的抗原决定簇存在于膜外区的N端(氨基酸3437,40,4245,5256),TSHR刺激阻断型抗体(TSBAb)的抗原决定簇存在于膜外区的C端(氨基酸301,385,390),而这些区域也都是TSH的结合位点。TSH主要作用的靶器官是甲状腺,它是通过与甲状腺细胞膜上的TSH受体TSHR结合,通过CAMP第二信号系统引起一系列生物学反应,促进甲状腺激素的分泌,提高甲状腺的摄碘能力并促进甲状腺激素的合成、释放,对甲状腺组织的生长、分化等起着非常重要的作用。TSHR属甲状腺的正常组分,但是,遗传因素的存在和免疫因素、环境因素的诱发,引起了hTSHR的变异,使其产生了抗原性,致TSHR特异性免疫原肽得以暴露,破坏了人体对TSHR的耐受性,产生大量的TRAb,引发免疫应答而致病。TRAb的检测在AITD的科研及临床中有着极其重要的意义。
目的
以本室制备富含TSAb抗原决定簇的重组TrxTSHRn蛋白、富含TSBAb抗原决定簇的重组TrxTSHRc蛋白分别作为抗原,酶联免疫吸附(ELISA)检测人血清TRAb(包括TSAb、TSBAb)含量,探讨此TRAb检测在AITD诊断和治疗中的意义。
方法
取本室研制的重组TrxTSHRn、TrxTSHRc蛋白为抗原,通过不同的抗原包被量、不同的血清稀释度优化,建立TSAb、TSBAb的间接ELISA检测方法。经精密度测试后,用于检测不同年龄、性别组健康人群,及各甲状腺疾病患者血清中的TRAb。
结果
1.以TrxTSHRc蛋白为抗原TRAb检测,抗原包被量16ng/100μl、待测血清稀释100倍时阳性和阴性血清OD405分别为1.325和0.358,批内变异cv%为3.77%和3.42%,批间变异cv%为4.87%和5.12%,正常人中OD405值为0.319±0.107,正常值范围为0.105～0.533,正常人组阳性率3.30%(9/272);Hashimoto甲状腺炎患者阳性率66.01%(336/509),其中伴甲状腺功能减退者阳性率77.78%(252/324)、伴甲状腺功能亢进者52.94%(63/119)、甲功正常者31.82%(21/66);初发Graves'患者阳性率41.86%(44/105);治疗1年Graves'患者阳性率17.86%(5/28);单纯性甲状腺肿患者阳性率6.98%(3/43);亚急性甲状腺炎患者阳性率11.1%(5/45);非毒性结节性甲状腺肿患者阳性率8.70%(6/69);甲状腺腺瘤患者阳性率3.2%(1/31)。
2.以TrxTSHRn蛋白为抗原TRAb检测,抗原包被量100ng/100μl、待测血清稀释100倍时阳性和阴性血清OD405分别为1.098和0.410,批内变异为3.53%和4.12%,批间变异为4.52%和4.67%,正常人群中儿童组共115例(0至14岁,平均3.8岁)OD405为0.186±0.094,正常成人组1060例(18至81岁)OD405值为0.398±0.167,二者差异有统计学意义,性别组之间及成人组各年龄段之间差异无统计学意义。成人组正常值范围0.064～0.732,阳性率2.45%(26/1060)、初发Graves'患者阳性率70.31%(74/105)、治疗1年Graves'患者阳性率35.7%(10/28)、Hashimoto甲状腺炎患者阳性率36.67%(44/120)、单纯性甲状腺肿患者阳性率4.6%(2/43)、亚急性甲状腺炎患者阳性率6.67%(3/45)、非毒性结节性甲状腺肿患者阳性率7.25%(5/69)。
结论
1.以TrxTSHRc蛋白为抗原ELISA法检测TRAb对AITD,尤其是Hashimoto甲状腺炎伴甲减的诊断中有重要意义。
2.以TrxTSHRn蛋白为抗原ELISA法检测TRAb值范围在儿童组,成人年组存在差异,有统计学意义,在男、女性别组差异无统计学意义。
3.以TrxTSHRn蛋白为抗原ELISA法检测TRAb对AITD,尤其是Graves'病的诊断中有重要意义。
Autoimmune thyroid disease(AITD)is a sort of organ specificity autoimmune disease.Human thyroid stimulating hormone receptor is a primary target antigen in AITD.And plays a role in set and process of AITD.The hTSHR gene assignment at No.14 chromatin body(14q31),is a G protein-coupled large moleculer glycoprotein consisting of extracellular(TSHR-ecd),transmembrane and intramembrane domain.TSHR-ecd has multi-discontinuous binding site where the thyrotropic-stimulating hormone(TSH)and thyrotrophin receptor antibody(TRAb) can discriminate and combine.Epitopes of thyroid-stimulating antibody(TSAb) mainly lie in N terminal of TSHR-ecd(amino acid3437,40,4245,5256),while thyroid stimulating blocking antibody(TSBAb)in C terminal(amino acid 301,385,390).TSH's target organ is thyroid gland.TSH can combin with TSHR and cause a series of biochemical event by CAMP second signal system.Physiologically, the combination can regulate the normal function and growth of thyroid follicular epithelial cell.TSHR is the normal composition of the thyroid gland.But under the function of heredity and environment,the structure of TSHR changes and has the antigenicity which can cause autoimmune thyroid disease(AITD)and product autoantibodies(TRAb).The detection of TRAb is very important for the dignosis, treatment and prognostic evaluation.
Objective:
Using Recombinent TrxTSHRn Protein which contains TSAb antigen determinant.and TrxTSHRc Protein which contains TSBAb antigen determinant as antigen,establish enzyme linked immunosorbent assay(ELISA).for detecting the TRAb in the serum of health and thyroid disease patient.Investigate this detection's significance for the dignosis,treatment in AITD。
Method:
Using Recombinent TrxTSHRn Protein and TrxTSHRc Protein as antigen, determin the optimum condition,establish the indirect ELISA for detecting the TRAb in the serum of health and thyroid disease patient.
Result:
1.The detection of TRAb using recombinent TrxTSHRc protein as antigen: It is the best condition for ELISA that the coating quantity of antigen is 16ng/100μl and the seru dilute 100 fold.The intra-assay coefficients of variation(CV)of positive seru and negtive seru are 3.5%,4.12%respectively.The interassay CV values are 4.52%,4.67%.The mean±standard deviation(SD)of normal value is 0.319±0.107(in adult).The positive rate of untreanted Graves' disease are 41.86%,treated Graves' disease are 7.86%,Hashimoto thyroiditis 66.01%(hypothyroidism77.78% hyperthyroidism52.94%),simple goiter are 6.98%,subacute thyroiditis are 11.1%Non-toxic nodular goiter;nontoxic nodular goiter are 8.7%thyroid adenoma are 3.1%respectively.
2.The detection of TRAb using recombinent TrxTSHRn protein as antigen: It is the best condition for ELISA that the coating quantity of antigen is 100ng/100μl and the seru dilute 100 fold.The intra-assay coefficients of variation(CV)of positive seru and negtive seru are 3.77%,3.42%respectively.The interassay CV values are 4.87%,5.12%.The mean±standard deviation(SD)of normal value is 0.186±0.094(in children),0.398±0.167(in adult).The positive rate of untreanted Graves' disease are 70.31%,treated Graves' disease are 35.7%,Hashimoto thyroiditis 36.67%,simple goiter are 4.6%,subacute thyroiditis are 6.67%Non-toxic nodular goiter;nontoxic nodular goiter are 7.25%respectively.
Conclusion:
1.The detection of TRAb using recombinent TrxTSHRc protein as antigen is very important for the dignosis of AITD,especially in Hashimoto thyroiditis complicate hypothyroidism;
2.The detection of TRAb using recombinent TrxTSHRn protein as antigen in normal:There is difference in range of normal values between children and adult,but no statistically significant between male and female;
3.The detection of TRAb using recombinent TrxTSHRn protein as antigen is very important for the dignosis of AITD,especially in Graves'disease.
目的
以本室制备富含TSAb抗原决定簇的重组TrxTSHRn蛋白、富含TSBAb抗原决定簇的重组TrxTSHRc蛋白分别作为抗原,酶联免疫吸附(ELISA)检测人血清TRAb(包括TSAb、TSBAb)含量,探讨此TRAb检测在AITD诊断和治疗中的意义。
方法
取本室研制的重组TrxTSHRn、TrxTSHRc蛋白为抗原,通过不同的抗原包被量、不同的血清稀释度优化,建立TSAb、TSBAb的间接ELISA检测方法。经精密度测试后,用于检测不同年龄、性别组健康人群,及各甲状腺疾病患者血清中的TRAb。
结果
1.以TrxTSHRc蛋白为抗原TRAb检测,抗原包被量16ng/100μl、待测血清稀释100倍时阳性和阴性血清OD405分别为1.325和0.358,批内变异cv%为3.77%和3.42%,批间变异cv%为4.87%和5.12%,正常人中OD405值为0.319±0.107,正常值范围为0.105～0.533,正常人组阳性率3.30%(9/272);Hashimoto甲状腺炎患者阳性率66.01%(336/509),其中伴甲状腺功能减退者阳性率77.78%(252/324)、伴甲状腺功能亢进者52.94%(63/119)、甲功正常者31.82%(21/66);初发Graves'患者阳性率41.86%(44/105);治疗1年Graves'患者阳性率17.86%(5/28);单纯性甲状腺肿患者阳性率6.98%(3/43);亚急性甲状腺炎患者阳性率11.1%(5/45);非毒性结节性甲状腺肿患者阳性率8.70%(6/69);甲状腺腺瘤患者阳性率3.2%(1/31)。
2.以TrxTSHRn蛋白为抗原TRAb检测,抗原包被量100ng/100μl、待测血清稀释100倍时阳性和阴性血清OD405分别为1.098和0.410,批内变异为3.53%和4.12%,批间变异为4.52%和4.67%,正常人群中儿童组共115例(0至14岁,平均3.8岁)OD405为0.186±0.094,正常成人组1060例(18至81岁)OD405值为0.398±0.167,二者差异有统计学意义,性别组之间及成人组各年龄段之间差异无统计学意义。成人组正常值范围0.064～0.732,阳性率2.45%(26/1060)、初发Graves'患者阳性率70.31%(74/105)、治疗1年Graves'患者阳性率35.7%(10/28)、Hashimoto甲状腺炎患者阳性率36.67%(44/120)、单纯性甲状腺肿患者阳性率4.6%(2/43)、亚急性甲状腺炎患者阳性率6.67%(3/45)、非毒性结节性甲状腺肿患者阳性率7.25%(5/69)。
结论
1.以TrxTSHRc蛋白为抗原ELISA法检测TRAb对AITD,尤其是Hashimoto甲状腺炎伴甲减的诊断中有重要意义。
2.以TrxTSHRn蛋白为抗原ELISA法检测TRAb值范围在儿童组,成人年组存在差异,有统计学意义,在男、女性别组差异无统计学意义。
3.以TrxTSHRn蛋白为抗原ELISA法检测TRAb对AITD,尤其是Graves'病的诊断中有重要意义。
Autoimmune thyroid disease(AITD)is a sort of organ specificity autoimmune disease.Human thyroid stimulating hormone receptor is a primary target antigen in AITD.And plays a role in set and process of AITD.The hTSHR gene assignment at No.14 chromatin body(14q31),is a G protein-coupled large moleculer glycoprotein consisting of extracellular(TSHR-ecd),transmembrane and intramembrane domain.TSHR-ecd has multi-discontinuous binding site where the thyrotropic-stimulating hormone(TSH)and thyrotrophin receptor antibody(TRAb) can discriminate and combine.Epitopes of thyroid-stimulating antibody(TSAb) mainly lie in N terminal of TSHR-ecd(amino acid3437,40,4245,5256),while thyroid stimulating blocking antibody(TSBAb)in C terminal(amino acid 301,385,390).TSH's target organ is thyroid gland.TSH can combin with TSHR and cause a series of biochemical event by CAMP second signal system.Physiologically, the combination can regulate the normal function and growth of thyroid follicular epithelial cell.TSHR is the normal composition of the thyroid gland.But under the function of heredity and environment,the structure of TSHR changes and has the antigenicity which can cause autoimmune thyroid disease(AITD)and product autoantibodies(TRAb).The detection of TRAb is very important for the dignosis, treatment and prognostic evaluation.
Objective:
Using Recombinent TrxTSHRn Protein which contains TSAb antigen determinant.and TrxTSHRc Protein which contains TSBAb antigen determinant as antigen,establish enzyme linked immunosorbent assay(ELISA).for detecting the TRAb in the serum of health and thyroid disease patient.Investigate this detection's significance for the dignosis,treatment in AITD。
Method:
Using Recombinent TrxTSHRn Protein and TrxTSHRc Protein as antigen, determin the optimum condition,establish the indirect ELISA for detecting the TRAb in the serum of health and thyroid disease patient.
Result:
1.The detection of TRAb using recombinent TrxTSHRc protein as antigen: It is the best condition for ELISA that the coating quantity of antigen is 16ng/100μl and the seru dilute 100 fold.The intra-assay coefficients of variation(CV)of positive seru and negtive seru are 3.5%,4.12%respectively.The interassay CV values are 4.52%,4.67%.The mean±standard deviation(SD)of normal value is 0.319±0.107(in adult).The positive rate of untreanted Graves' disease are 41.86%,treated Graves' disease are 7.86%,Hashimoto thyroiditis 66.01%(hypothyroidism77.78% hyperthyroidism52.94%),simple goiter are 6.98%,subacute thyroiditis are 11.1%Non-toxic nodular goiter;nontoxic nodular goiter are 8.7%thyroid adenoma are 3.1%respectively.
2.The detection of TRAb using recombinent TrxTSHRn protein as antigen: It is the best condition for ELISA that the coating quantity of antigen is 100ng/100μl and the seru dilute 100 fold.The intra-assay coefficients of variation(CV)of positive seru and negtive seru are 3.77%,3.42%respectively.The interassay CV values are 4.87%,5.12%.The mean±standard deviation(SD)of normal value is 0.186±0.094(in children),0.398±0.167(in adult).The positive rate of untreanted Graves' disease are 70.31%,treated Graves' disease are 35.7%,Hashimoto thyroiditis 36.67%,simple goiter are 4.6%,subacute thyroiditis are 6.67%Non-toxic nodular goiter;nontoxic nodular goiter are 7.25%respectively.
Conclusion:
1.The detection of TRAb using recombinent TrxTSHRc protein as antigen is very important for the dignosis of AITD,especially in Hashimoto thyroiditis complicate hypothyroidism;
2.The detection of TRAb using recombinent TrxTSHRn protein as antigen in normal:There is difference in range of normal values between children and adult,but no statistically significant between male and female;
3.The detection of TRAb using recombinent TrxTSHRn protein as antigen is very important for the dignosis of AITD,especially in Graves'disease.
引文
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[38]Chazenbalk GD,Pichurin P,Mclachlan SM,et al.A direct binding assay for thyrotropin receptor autoantibodies.Thyroid 1999,9(11):1057-1061.
[39]Marakimi M,Mori M.Identification of immunogenic regions in human thyrotropin receptor for immunoglobulin G of patients with Graves' disease.Biochem Biophys Res Commun,1990 Aug 31;171(1):512-518.
[40]Valse H,Nakashima M,Graves PN,Tomer Y,Morris JC,Davies TF.Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice:evidence for intramolecular epitope spreading.Endocrinology 1995 Oct;136(10):4415-23
[41]汤特,孙桂珍.ELISA法检测Graves氏病患者血清甲状腺刺激性抗体.天津医药.1993.21.(6)323-325
[42]Smith BR,Bolton J,Young S,et al.A new assay for thyrotropin receptor autoantibodies.Thyroid,2004,14:830-835.
[43]Kamijo K,Ishikawa K,Tanaka M.Clinical evaluation of 3rd generation assay for thyrotropin receptor antibodies:the M22-biotin-based ELISA initiated by Smith.Edocri J,2005,52:525-529.
[44]Cho BY,Kim WB,Chung JH,et al.High prevalence and little change in TSH receptor blocking antibody titres with thyroxine and antithyroid drug therapy in patients with nongoitrous autoimmune thyroiditis.Clin Endocrinol(Oxf),1995,43:465-471.
[45]Cho BY.et al.Clinical application of TSH receptor antibodies in thyroid disease.J Korean Med Sci,2002,17:293-301
[46]Wallaschofski H,Miehle K,Mayer A,et al.Prediction of remission orrelapse for Graves' hyperthyroidism by the combined determination of stimulating,blocking and binding TSH2receptor antibodies after thewithdrawal of antithyroid drug treatment.Horm Metab Res,2002,34:383-388.
[47]陈宁,汤特等TSBAb—ELISA法的建立与临床初步应用.标记免疫分析与临床.1999.(6).2:201-203.
[48]Hashimoto T.The guidelines for the diagnosis and monitoring of endocrine diseases(except of diabetes mellitus).Rinsho Byori,2003,51:592-594
[49]Wiersinga WM,Prummel MF.Pathogenesis of Graves' ophalmopathy current understanding.J Clin Endocrinol Metab,2001,86:501-503.
[50]Massart C,Orgiazzi J,Maugendre D.Clinical validity of a new commercial method for detection of TSH-receptor binding antibodies in sera from patients with Graves' disease treated with antithyroid drugs.Clin Chim Acta,2001,304:39-47.
[51]Orgiazzi J,Madec AM.Reduction of the risk of relapse after withdrawal of medical therapy for Graves' disease.Thyroid,2002,12:849-853
[52]贾卫国,魏松全,陈德才等.TRAb间接ELISA测定的建立.华西医学.2000,15:249-251.
[53]Catherine Massart,Jacqueline Gibassier,Franck Verite,et al.Use of Chinese hamster ovary cell lines transfected with cloned human thyrotropin receptor for the measurement of thyroid-stimulating antibodies:advantages and difficulties.Clinica Chimica Acta.2000,291:67-81.
[1]Schwarz-Lauer L,Chazenbalk GD,Mclachlan SM,et al.Evidence for a simplified view of autoantibody interactions with the thyrotropin receptor.Thyroid,2002,12:115-120.
[2]Kosugi S,Ban T,Kohn LD.Identification of thyroidstimulating antibodyspecific interaction sites in the N-terminal region of the thyrotropin receptor.Mol Endocrinol,1993,7:114-130.
[3]Corivilan B,Laurent B,Lecomte M,et al.Role of the cyclic adenosine 3'-5'monophosphate and monophosphatidy/inositol-Ca2+ cascade in mediting the effects of thyrotropin and iodide on hormone synthesis and secretion in human thyroid slices.J Clin Endocrinol Metab 1994,79:152-159.
[4]Li X,Lu S,Miyagi E,et al.Thyrotropin prevents apoptosis by promoting cell adhesion and cell cycle progression in FRTL25.[J].Endocrinology,1999,140:5970-5976.
[5]Khoo DH,Ens PH,H0 SC,et al.Graves' ophthalmopathy in the absence of elevated free thyroxine and tfiiodothyronine levels:prevalence,natural history,and thyrotropin receptor an tibody levels.Thyroid,2000,20:1093-1100.
[6]Wallaschofski H,Paschke R.at al.Detection of thyroid stimulating(TSAB)-and thyrotropin stimulation blocking(TSBAB)antibodies with CHO cell lines expressing different TSH-receptor numbers.Clin Endocrinol(Oxf).1999.50(3):365-72.
[7]Jordan NJ,Rinderle C,Ashfield J,Morgenthaler NG,Lazarus J,Ludgate M,Evans C.at al.A luminescent bioassay for thyroid blocking antibodies.Clin Endocrinol.2001.54(3):355-64.
[8]Kamijo K.TSH-receptor antibody measurement in patients with various thyrotoxicosis and Hashimoto's thyroiditis:a comparison of two two-step assays,coated plate ELISA using porcine TSH-receptor and coated tube radioassay using human recombinant TSH-receptor.Endocr J.2003 Feb;50(1):113-6.
[9]陈宁,汤特等TSBAb-ELISA法的建立与临床初步应用.标记免疫分析与临床.1999.(6).2:201-203.
[10] Moriyama K, Okuda J, Saijo M, Hattori Y, Kanamoto N, Hataya Y, Matsuda F, Mori T, Nakao K, Akamizu T.Recombinant monoclonal thyrotropin-stimulation blocking antibody (TSBAb) established from peripheral lymphocytes of a hypothyroid patient with primary myxedema. J Endocrinol Invest. 2003 Nov;26(11): 1076-80.
[11] Kamijo K, Ishikawa K,Tanaka M.Clinical evaluation of 3rd generation assay for thyrotropin receptor antibodies: the M22-biotin-based ELISA initiated by Smith. Edocri J,2005,52:525-529.
[12] Tada H, Izumi Y, Watanabe Y, Takano T, Fukata S, Kuma K, Hidaka Y, Amino N at al. Blocking type anti-tSH receptor antibodies detected by radioreceptor assay in Graves' disease. Endocr J. 2001 Dec;48(6):703-10.
[13] Tada H, Mizuta I, Takano T, Tatsumi KI, Izumi Y, Hidaka Y, Amino N. at al. Blocking-type anti-TSH receptor antibodies and relation to responsiveness to antithyroid drug therapy and remission in Graves' disease. [J]Clin Endocrinol (Oxf). 2003 Apr;58(4):403-8.
[14] Wallaschofski H, Miehle K, Mayer A, Tuschy U, Hentschel B, Paschke R.at al.Prediction of remission or relapse for Graves' hyperthyroidism by the combined determination of stimulating, blocking and binding TSH-receptor antibodies after the withdrawal of antithyroid drug treatment. Horm Metab Res. 2002 Jul;34(7):383-3888.
[15] Cho BY. Clinical application of TSH receptor antibodies in thyroid diseae. J Korean Med Sci, 2002, 17: 293-301.
[16] Amino N, Izumi Y, Hidaka Y, Takeoka K, Nakata Y, Tatsumi KI, Nagata A, Takano TNo increase of blocking type anti-thyrotropin receptor antibodies during pregnancy in patients with Graves' disease. J Clin Endocrinol Metab. 2003 Dec;88(12):5871-4.
[17] Kamijo K. at al.TSH-receptor antibodies determined by the first, second and third generation assays and thyroid-stimulating antibody in pregnant patients with Graves' disease. Endocr [J] 2007 Aug;54(4):619-24.
[18] Lugate M, Baker G. Unlocking the mechanisms of orbital inflammationin the thyroid eye disease[J]. Clin Exp Immunol, 2002 , 127 : 193-198.
[19] Kim WB, Chung HK, Park YJ, Park DJ, Lee HK, Cho BY.at al.Clinical significance of classification of Graves' disease according to the characteristics of TSH receptor antibodies.Korean[J]Intern Med.2001 Sep;16(3):187-200.
[20]黄晓东 顾学范 沈永年 张雅芬 叶军 陈瑞冠.先天性原发性甲状腺功能减低症50例TSHR基因研究临床儿科杂志.2007.25.7:562-564.
[21]Kosugi S,et at.Role of cysteine residues in the extrace llular domain and exoplasmic loops of the transmembrane domain of the TSH receptor.Biochem Biophys Res Commun,1992:189(3):1754.
[22]Takasu N,Yamada T,Takasu M,et al.Disappearance of thyrotrop in blocking antibodies and spontaneous recovery from hypothyroidism in autoimmune thyroiditis.N Engl J Med,1992,326:513-518.
[23]Vitti P,Chiovato L,Fiore E,et al.Use of cells expressing the human thyrotrop in(TSH)recep tor for the measurement of thyroid stimulating and TSH-blocking antibodies.ActaMed Austriaca,1996,23:52-56.
[24]Chiovato L,Vitti P,Santini F,et al.Incidence of antibodies blocking thyrotrop in effect in vitro in patients with euthyroid or hypothyroid autoimmune thyroiditis.J Clin EndocrinolMetab,1990,71:40-45.
[25]Cho BY,Kim WB,Chung JH,et al.High prevalence and little change in TSH recep tor blocking antibody titres with thyroxine and antithyroid drug therapy in patients with nongoitrous autoimmune thyroiditis.Clin Endocrinol (Oxf),1995,43:465-471.
[26]满娜,陈威,滕卫平,等.甲状腺功能减退症患者血清甲状腺刺激阻断性抗体随访研究.[J]中华内分泌代谢杂志,2005,21:114-117.
[27]Cho BY,et at.Transient neontal hypothyroidism due to transplac ental transfer of maternal im munogloblins that inhibit TSH binding;TSH induced cAMP increase and cell growth Endocrinol Jpn,1988;35:819.
[28]Fenzi G Subclinical hypothyroidism In the thyroid and age.[M]eds Pinchera A,Mann K,and Hoslalek U,Merck,New,York:schattaner stuttgart,1998.305-314.
[29]满娜,李玉姝,滕卫平,单忠艳,关海霞,范晨玲,毛金媛.亚临床甲状腺功能减退症患者的自身免疫特征及预后影响因素[J]中华内科杂志.2005.44(8):621-622.
[2] Nagayama Y, Kaufman KD, Seto P, Rapoport B. Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun . 1989,165:1184-1190.
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[6] Corivilan B, Laurent B, Lecomte M, et al. Role of the cyclic adenosine 3'-5' monophosphate and monophosphatidy/inositol-Ca~(2+) cascade in mediting the effects of thyrotropin and iodide on hormone synthesis and secretion in human thyroid slices. [J] Clin Endocrinol Metab 1994,79:152-159.
[7] Kim.W. B. Cho.B.Y. Park.H.Y. Lee, II.K.Kohn. LD. Tahara K and Koh ,C.S. Epitopes for thyroid-stimulating antibodies in graves' sera: a possible link of heterogenity to differences in response to antithyroid drug treatment.[J]Clin. Endocrinol. metab . 1996, 81:1758-1767.
[8] Sugawa H, Akamizu t, Kosugi S,Ueda Y, Ohta C . Okuda J .and Mori T. Precence of heterogenous thyroid -stimulating antibodies in sera from individual Graves patients as shown by synthesized thyrotropin receptor pepude , l31.c.lTevidence showing independent epitopes and a possible recognition of epitopic regiens by one antibody molecule. Eur. J. Endocrinol, 1995,133:283-293.
[9] Valse H, Nakashima M, Graves PN, Tomer Y, Morris JC, Davies TF. Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: evidence for intramolecular epitope spreading. Endocrinology 1995 Oct; 136(10): 4415-23.
[10] Ohmori M, Endo T, Ikeda M, Onaya T . Role of N-terminal region of the thyrotropin (TSH) receptor in signal transduction for TSH or thyroid stimulating antibody. Biochem Biophys Res Commun, 1991, 178:733-738.
[11] Kosugi S, Ban T, Kohn LD . Identification of thyroidstimulating antibody-specific interaction sites in the N-terminal region of the thyrotropin receptor. Mol Endocrinol, 1993, 7: 114-130.
[12] Schwarz-Lauer L, Chazenbalk GD , Mclachlan SM, et al. Evidence for a simplified view of autoantibody interactions with the thyrotropin receptor. Thyroid , 2002 , 12: 115-120.
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[15] Morgenthaler NG, Ho SC, Minich WB, at al. Stimulating and blocking thyroid-stimulating hormone (TSH) receptor autoantibodies from patients with Graves' disease and autoimmune hypothyroidism have very similar concentration, TSH receptor affinity, and binding sites. J Clin Endocrinol Metab. 2007 Mar;92(3): 1058-65.
[16] Quadbeck B, Hoermann R, Hahn S, at al.Binding, stimulating and blocking TSH receptor antibodies to the thyrotropin receptor as predictors of relapse of Graves' disease after withdrawal of antithyroid treatment. Horm Metab Res. 2005 Dec;37(12):745-50.
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[19] 方佩华,Graves病与自身免疫性抗体．[J]国外医学内分泌学分册,2003,23(6)：364—366．
[20] Bagriacik E.U, and Klein J.R. The thyrotropin (thyroid-stimulating hormone) receptor is expressed on murine dendritic cells and on a subset of CD45RBhigh lymph node T cells: functional role for thyroid-stimulating hormone during immune activation. J. Immunol. 2000,164:6158-6165.
[21] Spitzweg C, Joba W, and Heufelder A.E. Expression of thyroid-related genes in human thymus.Thyroid. 1999,9:133-141.
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[23] Brokken L.J, Scheenhart J.W, Wiersinga W.M,and Prummel M.F. Suppression of serumTSH by Graves' Ig: evidence for a functional pituitaryTSH receptor. [J]. Clin. Endocrinol. Metab. 2001,86:4814-4817.
[24] Lugate M, Baker G. Unlocking the mechanisms of orbital inflammationin the thyroid eye disease. [J]. Clin Exp Immunol, 2002 , 127 : 193-198
[25] Mojor BJ , Curres A. The full length and solice variant thyrotropin receptor is expressed exclusively in skeletal muscle of extraocular orgin : a link to the pathogenesis of Graves'ophthalmopathy[J] .Biochem Biophys ResCommun , 1997 ,7 : 493-496.
[26] Bahn RS , Dotton CM, Natt N , et al. Thyrotropin receptor expression in Graves'orbital adiposePconnective tissues : potential autoantigen in Graves' ophth almopathy [J]. J Clin Endocrinol Metab ,1998 ,83 :998-1002.
[27] Takasu N, Yamada T, Takasu M, et al. Disappearance of thyrotrop in blocking antibodies and spontaneous recovery from hypothyroidism in autoimmune thyroiditis. N Engl J Med, 1992, 326: 513-518.
[28] Vitti P, Chiovato L, Fiore E, et al. Use of cells exp ressing the human thyrotrop in (TSH) recep tor for the measurement of thyroid stimulating and TSH -blocking antibodies.ActaMed Austriaca,1996,23:52-56.
[29]Chiovato L,Vitti P,Santini F,et al.Incidence of antibodies blocking thyrotrop in effect in vitro in patients with euthyroid or hypothyroid autoimmune thyroiditis.J Clin EndocrinolMetab,1990,71:40-45.
[30]马绍刚,方佩华,杨箐岩,刘戈力,马成成,许静,李宁,陈慧,冯洁,TSH受体基因突变致先天性甲状腺功能减退症一例及其家系分析.[J]中华内分泌代谢杂志.2006,(22),1:41-44.
[31]Wallaschofski H,Kuwert T,Lohrnann T.at al.TSH-receptor autoantibodiesdifferentiation of hyperthyroidism between Graves' disease and toxic multinodular goitre.Exp Clin Endocrinol Diabetes.2004.112(4):171-4.
[32]Costagliola S,at al.Second filial generation TSH receptor antibody has the mrre sensitivity in Graves disease.[J]Clin Endocrinol Metab.1999,84.290-297.
[33]Pedersen IB,Knudsen N,Perrild H,Ovesen L,Laurberg P.TSH-receptor antibody measurement for differentiation of hyperthyroidism into Graves' disease and multinodular toxic goitre:a comparison of two competitive binding assays.Clin Endocrinol(Oxf).2001.55(3):381-90.
[34]Villalta D,Orunesu E,Tozzoli R,et al.Analytical and diagnostic accuracy of "second generation" assays for thyrotrophin receptor antibodies with radioactive and chemiluminescent tracers.J Clin Pathol,2004,57:378-382.
[35]罗国春,朱开思,汪寅章.用中国仓鼠卵巢细胞测定甲状腺刺激性抗体实验条件的探讨.[J]现代医学.2006.12.(34卷)6:400-402
[36]Wallaschofski H,Paschke R.at al.Detection of thyroid stimulating (TSAB)-and thyrotropin stimulation blocking(TSBAB)antibodies with CHO cell lines expressing different TSH-receptor numbers.Clin Endocrinol(Oxf).1999.50(3):365-72.
[37]Jordan NJ,Rinderle C,Ashfield J,Morgenthaler NG,Lazarus J,Ludgate M,Evans C.at al.A luminescent bioassay for thyroid blocking antibodies.Clin Endocrinol.2001.54(3):355-64.
[38]Chazenbalk GD,Pichurin P,Mclachlan SM,et al.A direct binding assay for thyrotropin receptor autoantibodies.Thyroid 1999,9(11):1057-1061.
[39]Marakimi M,Mori M.Identification of immunogenic regions in human thyrotropin receptor for immunoglobulin G of patients with Graves' disease.Biochem Biophys Res Commun,1990 Aug 31;171(1):512-518.
[40]Valse H,Nakashima M,Graves PN,Tomer Y,Morris JC,Davies TF.Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice:evidence for intramolecular epitope spreading.Endocrinology 1995 Oct;136(10):4415-23
[41]汤特,孙桂珍.ELISA法检测Graves氏病患者血清甲状腺刺激性抗体.天津医药.1993.21.(6)323-325
[42]Smith BR,Bolton J,Young S,et al.A new assay for thyrotropin receptor autoantibodies.Thyroid,2004,14:830-835.
[43]Kamijo K,Ishikawa K,Tanaka M.Clinical evaluation of 3rd generation assay for thyrotropin receptor antibodies:the M22-biotin-based ELISA initiated by Smith.Edocri J,2005,52:525-529.
[44]Cho BY,Kim WB,Chung JH,et al.High prevalence and little change in TSH receptor blocking antibody titres with thyroxine and antithyroid drug therapy in patients with nongoitrous autoimmune thyroiditis.Clin Endocrinol(Oxf),1995,43:465-471.
[45]Cho BY.et al.Clinical application of TSH receptor antibodies in thyroid disease.J Korean Med Sci,2002,17:293-301
[46]Wallaschofski H,Miehle K,Mayer A,et al.Prediction of remission orrelapse for Graves' hyperthyroidism by the combined determination of stimulating,blocking and binding TSH2receptor antibodies after thewithdrawal of antithyroid drug treatment.Horm Metab Res,2002,34:383-388.
[47]陈宁,汤特等TSBAb—ELISA法的建立与临床初步应用.标记免疫分析与临床.1999.(6).2:201-203.
[48]Hashimoto T.The guidelines for the diagnosis and monitoring of endocrine diseases(except of diabetes mellitus).Rinsho Byori,2003,51:592-594
[49]Wiersinga WM,Prummel MF.Pathogenesis of Graves' ophalmopathy current understanding.J Clin Endocrinol Metab,2001,86:501-503.
[50]Massart C,Orgiazzi J,Maugendre D.Clinical validity of a new commercial method for detection of TSH-receptor binding antibodies in sera from patients with Graves' disease treated with antithyroid drugs.Clin Chim Acta,2001,304:39-47.
[51]Orgiazzi J,Madec AM.Reduction of the risk of relapse after withdrawal of medical therapy for Graves' disease.Thyroid,2002,12:849-853
[52]贾卫国,魏松全,陈德才等.TRAb间接ELISA测定的建立.华西医学.2000,15:249-251.
[53]Catherine Massart,Jacqueline Gibassier,Franck Verite,et al.Use of Chinese hamster ovary cell lines transfected with cloned human thyrotropin receptor for the measurement of thyroid-stimulating antibodies:advantages and difficulties.Clinica Chimica Acta.2000,291:67-81.
[1]Schwarz-Lauer L,Chazenbalk GD,Mclachlan SM,et al.Evidence for a simplified view of autoantibody interactions with the thyrotropin receptor.Thyroid,2002,12:115-120.
[2]Kosugi S,Ban T,Kohn LD.Identification of thyroidstimulating antibodyspecific interaction sites in the N-terminal region of the thyrotropin receptor.Mol Endocrinol,1993,7:114-130.
[3]Corivilan B,Laurent B,Lecomte M,et al.Role of the cyclic adenosine 3'-5'monophosphate and monophosphatidy/inositol-Ca2+ cascade in mediting the effects of thyrotropin and iodide on hormone synthesis and secretion in human thyroid slices.J Clin Endocrinol Metab 1994,79:152-159.
[4]Li X,Lu S,Miyagi E,et al.Thyrotropin prevents apoptosis by promoting cell adhesion and cell cycle progression in FRTL25.[J].Endocrinology,1999,140:5970-5976.
[5]Khoo DH,Ens PH,H0 SC,et al.Graves' ophthalmopathy in the absence of elevated free thyroxine and tfiiodothyronine levels:prevalence,natural history,and thyrotropin receptor an tibody levels.Thyroid,2000,20:1093-1100.
[6]Wallaschofski H,Paschke R.at al.Detection of thyroid stimulating(TSAB)-and thyrotropin stimulation blocking(TSBAB)antibodies with CHO cell lines expressing different TSH-receptor numbers.Clin Endocrinol(Oxf).1999.50(3):365-72.
[7]Jordan NJ,Rinderle C,Ashfield J,Morgenthaler NG,Lazarus J,Ludgate M,Evans C.at al.A luminescent bioassay for thyroid blocking antibodies.Clin Endocrinol.2001.54(3):355-64.
[8]Kamijo K.TSH-receptor antibody measurement in patients with various thyrotoxicosis and Hashimoto's thyroiditis:a comparison of two two-step assays,coated plate ELISA using porcine TSH-receptor and coated tube radioassay using human recombinant TSH-receptor.Endocr J.2003 Feb;50(1):113-6.
[9]陈宁,汤特等TSBAb-ELISA法的建立与临床初步应用.标记免疫分析与临床.1999.(6).2:201-203.
[10] Moriyama K, Okuda J, Saijo M, Hattori Y, Kanamoto N, Hataya Y, Matsuda F, Mori T, Nakao K, Akamizu T.Recombinant monoclonal thyrotropin-stimulation blocking antibody (TSBAb) established from peripheral lymphocytes of a hypothyroid patient with primary myxedema. J Endocrinol Invest. 2003 Nov;26(11): 1076-80.
[11] Kamijo K, Ishikawa K,Tanaka M.Clinical evaluation of 3rd generation assay for thyrotropin receptor antibodies: the M22-biotin-based ELISA initiated by Smith. Edocri J,2005,52:525-529.
[12] Tada H, Izumi Y, Watanabe Y, Takano T, Fukata S, Kuma K, Hidaka Y, Amino N at al. Blocking type anti-tSH receptor antibodies detected by radioreceptor assay in Graves' disease. Endocr J. 2001 Dec;48(6):703-10.
[13] Tada H, Mizuta I, Takano T, Tatsumi KI, Izumi Y, Hidaka Y, Amino N. at al. Blocking-type anti-TSH receptor antibodies and relation to responsiveness to antithyroid drug therapy and remission in Graves' disease. [J]Clin Endocrinol (Oxf). 2003 Apr;58(4):403-8.
[14] Wallaschofski H, Miehle K, Mayer A, Tuschy U, Hentschel B, Paschke R.at al.Prediction of remission or relapse for Graves' hyperthyroidism by the combined determination of stimulating, blocking and binding TSH-receptor antibodies after the withdrawal of antithyroid drug treatment. Horm Metab Res. 2002 Jul;34(7):383-3888.
[15] Cho BY. Clinical application of TSH receptor antibodies in thyroid diseae. J Korean Med Sci, 2002, 17: 293-301.
[16] Amino N, Izumi Y, Hidaka Y, Takeoka K, Nakata Y, Tatsumi KI, Nagata A, Takano TNo increase of blocking type anti-thyrotropin receptor antibodies during pregnancy in patients with Graves' disease. J Clin Endocrinol Metab. 2003 Dec;88(12):5871-4.
[17] Kamijo K. at al.TSH-receptor antibodies determined by the first, second and third generation assays and thyroid-stimulating antibody in pregnant patients with Graves' disease. Endocr [J] 2007 Aug;54(4):619-24.
[18] Lugate M, Baker G. Unlocking the mechanisms of orbital inflammationin the thyroid eye disease[J]. Clin Exp Immunol, 2002 , 127 : 193-198.
[19] Kim WB, Chung HK, Park YJ, Park DJ, Lee HK, Cho BY.at al.Clinical significance of classification of Graves' disease according to the characteristics of TSH receptor antibodies.Korean[J]Intern Med.2001 Sep;16(3):187-200.
[20]黄晓东 顾学范 沈永年 张雅芬 叶军 陈瑞冠.先天性原发性甲状腺功能减低症50例TSHR基因研究临床儿科杂志.2007.25.7:562-564.
[21]Kosugi S,et at.Role of cysteine residues in the extrace llular domain and exoplasmic loops of the transmembrane domain of the TSH receptor.Biochem Biophys Res Commun,1992:189(3):1754.
[22]Takasu N,Yamada T,Takasu M,et al.Disappearance of thyrotrop in blocking antibodies and spontaneous recovery from hypothyroidism in autoimmune thyroiditis.N Engl J Med,1992,326:513-518.
[23]Vitti P,Chiovato L,Fiore E,et al.Use of cells expressing the human thyrotrop in(TSH)recep tor for the measurement of thyroid stimulating and TSH-blocking antibodies.ActaMed Austriaca,1996,23:52-56.
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