The Establishment And Clinical Application Of Quantitative Fluorescent-Enzyme Immunoassay For The Measurement Of Human Thyrotropin Receptor Antibody

Autoimmune thyroid disease(AITD) includes Graves'disease(GD) and Hashimoto's thyroiditis(HT). Thyrotropin receptors(TSHR) localize on thyroid follicular epithelial cell plasma membranes, which are involved in the normal regulation of thyroid fundation and in the pathology of AITD. TSHR has been known as the most important autoantigen for AITD which stimulates body to generate autoantibodies(TRAb), including thyroid stimulating antibody(TSAb) resulting in GD and thyroid stimulating blocking antibody (TSBAb) leading to HT. TSHR extracellular domain (TSHR-ecd) is the major sites for binding with TSH and TRAb. TSHR-ecd amino terminus(N-terminal) are the major epitopes for binding with TSAb. Our laboratory recombined human TSHR-ecd amino terminus gene(462bP), and obtained TSHR-ecd amino terminus fusion protein(TrxFus-hTSHRn) by prokaryotic expression in E.Coli BL21 StarTM(DE3).Fluorescent-enzyme immunoassay (FEIA) is a sensitive method to detect trace materials based on enzyme-linked immunoserbent assay(ELISA). The aim of this study was to establish a quantitative FEIA to measure human thyroid stimulating antibody(TSAb)which could be used in diagnosis, treatment and prognostic evaluation for autoimmune thyroid disease (AITD). Objective:To establish a quantitative human serum TSAb FEIA using recombination hTSHR-ecd amino terminus fusion proteins(TrxFus-hTSHRn) as antigen, in which could be used in clinical diagnosis and monitoring for AITD patients.Methods:The recombinant plasmid pET102/D-hTSHRn462bP encoding hTSHR-ecd amino terminus fusion protein (TrxFus-hTSHRn) was expressed in E.Coli BL21 StarTM(DE3).Then,the purified TrxFus-hTSHRn fusion protein was assessed. The FEIA detecting hTSAb using TrxFus-hTSHRn as the antigen was established, hTSAb in serum as the first antibody, goat anti-human IgG labelled AP as the second antibody,4-MUP as the fluorescent substrate, the home-made TSAb calibration materials were calibrated against the WHO standard NIBSC 90/672. The TSAb FEIA was applied to thyroid disease patients. The cut-off point value was determined with 167 young men.559 serum of thyroid disease patients were measured, including 183 cases of untreated Graves'disease with hyperthyroidism,56 cases of Graves'disease treated in 3-9 months,64 cases of Graves'disease treated in 1 year,112 cases of Hashimoto's thyroiditis with hypothyroidism,22 cases of simple goiter,49 cases of nontoxic nodular goiter,21 cases of thyroid adenoma,52 cases of subacute thyroiditis.Results:1.The E.Coli BL21 StarTM(DE3) was selected as the expressing strain. Induced by IPTG with the concentration of 1mM in 37℃for 4 hours. The expressed protein was purified with Ni2+-NTA by affinity chromatography and TrxFus-hTSHRn with satisfactory immune activity was obtained. The molecular mass of TrxFus-hTSHRn was 37.5KD with 91.2% in purity. The production rates of TrxFus-hTSHRn were 21.5-28.4mg/L medium and its satisfactory antigen activity was identified by Western Blotting.2.The optimum conditions of FEIA:The optimum antigen coating dose was 100ng/well. The dilution of patients'serum and second antibody were 1:100 and 1:20000 respectively. The optimum temperature and time for every step after coating were 37℃and 60 minutes. Finally, adding 50mM EDTA to stop the reaction.3.The assessment for the TSAb FEIA:Sensitivity:0.05mIU/L. Specificity: TrxFus-hTSHRn specific binding with hTRAb and there was no cross-reaction between TrxFus-hTSHRn and hTPOAb. Precision:the intra-assay coefficient of variation (CV) of positive and negative serum were 4.47% and 9.25%; the inter-assay CV were7.29% and 13.57%. Accuracy:sample recovery rates were 102.13%,97.50 %,98.00% for high, medium and low samples, respectively.Validity:dilution test showed acceptable validity, as the diluted curve run nearly parallel to the standard curve. Correlation:the correlation coefficient between FEIA and commercial kit was well (r=0.767,P<0.05; kappa=0.791,P<0.01).4.Cut-off point value:measuring TSAb of 167 young men, the TSAb concentrations(x+2s) above 42.9 mlU/L are considered to be positive. 5.Clinical application:the TSAb concentrations were 142.5±23.2 mIU/L and the positive ratio was 88.94% in untreated Graves'disease with hyperthyroidism; 93.1±30.3 mIU/L and 64.09% in Graves'disease treated in 3-9 months; 75.6±35.0 mIU/L and 38.62% in Graves'disease treated in 1 year; 71.3±14.7 mlU/L and 61.88% in Hashimoto's thyroiditis with hypothyroidism; 25.6±12.0 mIU/L and 0.85% in simple goiter; 38.7±13.5 mIU/L and 3.71% in nontoxic nodular goiter; 41.0±8.8 mlU/L and 5.84%in thyroid adenoma; 40.3±10.6 mIU/L and 11.69% in subacute thyroiditis.The results of untreated Graves'disease with hyperthyroidism patients showed significant difference compared with other groups(P<0.01).Conclusion:l.hTSHRn could be expressed effectually in the E.Coli BL21 StarTM(DE3). The production rates of TrxFus-hTSHRn were 21.5-28.4mg/L medium and its satisfactory antigen activity was identified by Western Blotting.2.The quantitative TSAb FEIA was established using recombinant TrxFus-hTSHRn. This method showed specificity, precision, accuracy, stability and satisfactory correlation with commercial kit.3.The method was applied in thyroid disease patients. The positive rate of Graves'disease was highest. Therefore, the FEIA was an effective method of diagnosis in Graves'disease and distinguishing AITD and non-AITD.