重组人TSHR片段蛋白建立人TRAb ELISA及临床应用

A novel human TSHR antibody ELISA using recombinant extracellular domain fragments of human TSH receptor as antigen and initial clinical evaluation

目的建立2种检测人血清促甲状腺激素受体抗体（TRAb）酶联免疫吸附测定（ELISA）方法，分别以测定刺激性抗体（TSAb）和阻断性抗体（TSBAb）为主，并用于甲状腺疾病检测。方法分别以重组人促甲状腺激素受体（TSHR）膜外区氨基端（N端）蛋白和羧基端（C端）蛋白作为抗原建立N法和C法，各检测健康人229名，初诊Graves病（GD）451例、初诊慢性淋巴细胞性甲状腺炎（HT）伴甲状腺功能减退症（简称甲减）104例、单纯性甲状腺肿35例、非毒性结节性甲状腺肿47例、亚急性甲状腺炎38例。组间比较采用方差（ANOVA）分析，组间阳性率比较用χ^2检验。结果N法：（1）与商品TSAb试剂盒检测法明显相关（r=0．7422，P〈0．05）。（2）批内、批间CV分别为3．96％～4．67％和7．32％～8．33％。（3）健康人405nm处吸光度（A405）值为0．44±0．15，阳性切点（x＋2s）为0．74。（4）初诊GD A405为0．93±0．28，高于健康人（F=2．0341，P=0．001），阳性率82．8％。（5）初诊HT伴甲减A405，为0．64±0．23，高于健康人（F=1．7822，P=0．003），阳性率62．1％。C法：（1）批内、批间CV分别为3．24％～4．72％和7．22％-8．55％。（2）健康人A405为0．56±0．11，阳性切点（x＋2s）为0．78。（3）初诊HT伴甲减A405为1．17±0．26，明显高于健康人（F=3．0333，P=0．002），阳性率93．4％。（4）初诊GD A405；为0．74±0．23，明显高于健康人（F=1．8339，P=0．03），阳性率66．3％。N法、C法检测单纯性甲状腺肿、非毒性结节性甲状腺肿、亚急性甲状腺炎患者与健康人差异均无统计学意义（N法：F=1．6134，1．5772和1．6208，C法：F=1．5910，1．5369和1．6055，P均〉0．05）。结论N法以检测人血清TSAb为主，C法以检测TSBAb为主；其为临床GD诊断、HT辅助诊断、疗效观察提供了新型、特异、稳定、可靠、操作简便的方法。

Objective Thyrotropin receptor antibodies （TRAb） include thyroid stimulating antibody （TSAb） and thyroid stimulating blocking antibody （TSBAb）. It is very helpful to detect serum TSAb and TSBAb levels in patients with thyroid diseases. The aim of this study was to establish two enzyme linked immunosorbent assay （ELISA） methods （N method and C method） that may determine serum TSAb and TSBAb levels respectively. The initial clinical application in thyroid diseases was also investigated. Methods Recombinant human thyroid stimulating hormone receptor （TSHR）-ecd N-terminal fragment was used in N method and C-terminal fragment was used in C method as antigens respectively. Two ELISA methods for detection of TSAb and TSBAb were established and evaluated. The absorbance value A405 was used as the quantitative parameter to reflect the level of TRAb. The serum TSAb and TSBAb levels were measured in 229 healthy controls, and 675 patients. They were 451 untreated Graves disease （GD）, 104 untreated chronic lymphocytic thyroiditis （Hashimoto disease, HT） complicating hypothyroidism, 35 simple goiters, 47 nodular goiters and 38 subacute thyroiditis. The ANOVA and χ^2-test were used for statistic data analysis. Results N method ： （ 1 ） the serum TRAb levels measured with N method were positively correlated with commercial TSAb kit （ r = 0. 7422, P 〈 0.05 ）. （ 2 ） The intra-assay coefficient of variation （CV） and the inter-assay CV were 3.96%- 4.67% and 7.32%- 8.33% , respectively. （3） A405 （ x ± s） value in healthy group was 0.44 ±0. 15 with a cut-off value （x＋2s） of 0.74. （4） In untreated GD group, A405 was 0.93 ±0.28, which was significantly higher than that in healthy group （ F = 2. 0341, P = 0. 001 ）. The positive rate was 82. 8%. （5） In untreated HT complicating hypothyroidism group, A405 was 0. 64 ±0. 23, which was significantly higher than that in healthy group （ F = 1. 7822, P = 0. 003 ）. The positive rate was 62. 1%. C method：（ 1 ） intra-assay CV and inter-assay CV were 3.24%- 4.72% and 7.22%- 8.55% , respectively. （2） The measured A405 value in healthy group was 0.56 ± 0.11 with a cut-off value （x ＋ 2s） of 0.78. （3） In untreated HT complicating hypothyroidism group, A405 （x ＋ s） was 1. 17 ± 0.26, which was significantly higher than that in healthy group （F = 3. 0333, P = 0. 002）. The positive rate was 93.4%. （4） In untreated GD group, A405 was 0.74 ± 0.23, which was significantly higher than that in healthy group （F = 1. 8339, P = 0.03）. The positive rate was 66.3%. There were no significant differences among simple goiter, nodular goiter, subaeute thyroiditis and healthy groups （N method： F = 1. 6134, 1. 5772, 1. 6208, C method： F = 1. 5910, 1. 5369, 1. 6055, all P 〉0.05） assayed with both N method and C methods. Conclusions The present novel N method may mainly measure TSAb level and C method mainly measure TSBAb levels. The two ELISA methods showed to be accurate, stable and convenient. It may be helpful for clinical evaluation in GD, HT and other thyroid diseases.