The Identification Of Recombinant TrxTSHRn Protein, The Establishment Of ELISA For Detecting TRAb And Its Preliminary Application

Human thyrotropin receptor (hTSHR) is a kind of membrane protein which localize on thyroid follicular epithelial cell(TEC) plasma membranes. Recently TSHR was found on some others cells, such as lymphocyte, thymocyte, pituicyte, et al. Physiologically, the combination of TSHR and TSH can regulate the normal function and growth of TEC. 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). TRAb is a kind of polyclonal antibody including thyroid stimulating antibody(TSAb) leading to Graves'disease and thyroid stimulating blocking antibody (TSBAb) leading to Hashimoto disease. The detection of TRAb is very important for the dignosis, treatment and prognostic evaluation. TSHR is a seven-transmembrane, G protein-coupled large moleculer glycoprotein consisting of extracellular(TSHR-ecd), transmembrane and intramembrane domain. Major epitopes of TRAb are located at TSHR-ecd composed by 418 amino acid including 21 amino acid signal peptide. Epitopes of TSAb mainly lie in N terminal of TSHR-ecd while TSBAb in C terminal. TSHR-ecd is essential for binding with TSH, while transmembrane and intramembrane domain play an important role of transforming signal and combining with G protein.Objective: In this study, we plan to identify the molecular weight and immunocompetence of TSHR-ecd N terminal protein fused thioredoxin(TrxTSHRn) which expressioned by E.coli BL21 StarTM(DE3) and establish ELISA for Detecting TRAb Using Recombinent TrxTSHRn Protein.Method: 1. SDS-PAGE is used to identify the molecular weight of TrxTSHRn. Theimmunocompetence of TrxTSHRn is identified by the method of Western blot.2. TrxTSHRn is used as antigen to establish the indirect ELISA for detecting TRAb. Then apply the assay to detecting TRAb in patient of untreanted Graves' disease, Hashimoto thyroiditis, treated Graves' disease, nodular goiter.Result:1. The molecular weight of TrxTSHRn is 3 5.9kD by the method of SDS-PAGE. There is a difference of 4.36% between metered value and theoyical value.2. There is a dyeing strap in nitrocellulose filter when TRAb positive seru is used as first antibody, while no dyeing strap when TRAb negtive seru is used.3. It is the best condition for ELISA that the coating quantity of antigen is lOng/lOOy 1 and the seru dilute 200 fold. The intra-assay coefficients of variation (CV) of positive seru and negtive seru are 3.8%, 3.6% respectively. The interassay CV values are 5.15%, 3.93%. The mean±standard deviation(SD) of normal value is 0.46+0.15. The positive rate of untreanted Graves' disease, Hashimoto thyroiditis, treated Graves' disease are 70.4%, 40%, 36.6% respectively.Conclusion:1. The molecular weight of TrxTSHRn is 35.9kD and TrxTSHRn has immunocompetence.2. The indirect ELISA for detecting TRAb has been established and applied to clinic. The precision and sensitivity of this assay has been proved good.