| Fluorescent-Enzyme immunoassay (FEIA) was a more sensitive method to detect the trace based on the Enzyme-Linked Immunoserbent Assay(ELISA).Using FEIA,we tried to establish a quantitative assay to measure human Thyroid Peroxidase autoantibody (hTPOAb),which would be used in the diagnosis of autoimmune thyroid disease (AITD).Objective:Using the recombinant extracellular region proteins of hTPO as the antigen, a quantitative FEIA was established, which would be used in the clinical diagnosis and effect monitoring.Methods:The recombinant extracellular region protein, which contained the recombinant plasmid pGESJ including hTPO epitopes (gene 1702～2622, encoding A568-874), was expressed in the E. Coli BL21.Then, the purified GST-hTPO fusion protein was assessed. Afterwards, the FEIA to detect hTPOAb with GST-hTPO fusion protein as the antigen was established, hTPOAb in the sera as the first antibody, goat anti-human IgG labelled AP as the second antibody,4-MUP as the fluorescent substrate, the sera calibrated by the national reference as the standard substance. After appraisal, the method was applied to test the antibody in the sera. The normal value was decided by 262 young men.534 patients'seras were detected, including 270 cases of Graves' disease,166 cases of Hashimoto's thyroiditis,43 cases of simple goiter,38 cases of subacute thyroiditis and 17 cases of adenoma.Results:1 The E. Coli BL21 was selected as the expressing strain and IPTG (isopropylβ-D-thoiglactoside) as inducer with the concentration of 0.1 mM. GST-hTPO fusion protein with satisfactory immune activity was obtained after purification. The optimum temperature and time were 25℃and 5.5 hours. The expressed protein was purified with Glutathione Sepharose 4B by affinity chromatography. The molecular mass of GST-hTPO was 61.24 KD. The producing rate of GST-hTPO fusion protein was 12mg/L medium and its satisfactory antigen activity was verified by FEIA. After six months' observation with ELISA, the inter-assay coefficient of variation (CV) of the negative and the positive seras were 12.30% and 6.78% with the coated refrigerant strips. 2 The optimum condition of FEIA:The optimum antigen coating dose was 1μg/well. The dilution of patients' sera and second antiantibody were 1:100 and 1:20000 respectively; The optimum temperature and time for every step after coating were 37℃and 60 minutes. After adding the stop buffer EDTA, the reaction was balanced.The appraisal of FEIA:There was no cross-reaction between GST-hTPO fusion protein and Tg (Thyroglobulin). The intra-assay CV was 5.14%,19.36% and inter-assay CV was 6.72%,17.90% for the positive and negative seras. Sample recovery test's results showed 102%,92%,100% respectively for high, medium and low samples. Dilution test showed acceptable validity, as the diluted curve run nearly parallel to the standard curve. The correlation between FEIA and commodity kits was well (R=0.80, P<0.01).3 Clinical application:After testing 262 young men, titer above 5IU/mL was considered to be positive. The concentration of hTPOAb was showed in the form of medium.The results were 11.5 IU/ml and the positive ratio was 89.76% in HT; 4 IU/ml and 32.35% in pretherapy GD; 4 IU/ml and 31.68% in undergoing drug treatment GD;4 IU/ml and 26.32% in subacute thyroiditis; 3 IU/ml and zero in simple goiter; 4 IU/ml and 11.76% in adenoma. The results of Hashimoto's thyroiditis patients showed significant difference compared with other groups(p<0.01).Conclusion:1 hTPO could be expressed effectually in the E.coli BL21, the producing rate of GST-hTPO fusion protein was 12mg/L medium and the concentration was 2mg/ml2 After our standard substance was assured, the quantitative FEIA were established using recombinant fusion protein. This method showed specificity, precision, accuracy, stability and satisfactory correlation with the commodity kits.3 The method was applied in the serous measurement of thyroid disease patients. The positive rate of HT was the highest. Therefore, the FEIA was an effective method in diagnosing HT and distinguishing the AITD and non-AITD. |
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