Establishment Of An Indirect ELISA Using Human Recombinant Thyroglobulin To Detect TgAb And Its Preliminary Clinical Application

Autoimmune thyroid disease(AITD)is common in clinic.It is a group of disorders caused by autoaggression of the immune system,comprising of Graves disease,Hashimoto thyroditis and Idiopathic hypothyroidism.The autoantibody of human thyroglobulin(hTg)in sera is one of an index of immune state.So far,in TgAb assays used at home and abroad,the source of hTg standard is limited because it is extracted from human thyroid tissues and it may mix with many other proteins. Up to now,there is no report about the TgAb assay using the recombinant hTg as antigen.Objective:The hTg epitope(1491 bp)was expressed in prokaryotic system using molecular biology techniques and obtained the recombinant hTg fusion protein. Indirect enzyme-linked immunosorbent assay(ELISA)was constructed using the recombinant hTg as antigen.After this ELISA was identified,TgAb from 300 health controls were measured and the cut-off value was obtained.TgAb from 484 specimens in five kinds of thyroid diseases were measured,and then analyzed the statistic differences among them and the positive rates.Method:The recombinant prokaryotic expressive plasmid pET102/D-TOPO contained hTg epitope was extracted from E.coli TOP10 and transformed into E.coli BL21 Star^TM(DE3).The expressive condition of E.coli BL21 Star^TM(DE3)was at 25℃6 hours induced by 1.0mM isopropylβ-D-thioglactoside(IPTG).The bacteria were broken up by supersonic and the recombinant hTg was purified by Ni-NTA His·Bind resin.The molecule weight,immune activity and quantity of it was identified by SDS-PAGE,ELISA and the method of Comasse Brilliant blue, respectively.The ELISA for TgAb was established using recombinant hTg as antigen. The best working concentrations of TgAb antigen and sera specimens were determined by crossed serial dilution method.The inter-assay coefficient of variation (CV)and intra-assay CV was obtained.30 specimens were measured using RIA and ELISA assay,respectively.The correlation between two methods was obtained. TgAb from 300 healthy specimens were measured using ELISA and the cut-off value was obtained.TgAb from 484 specimens in five kinds of thyroid diseases were measured,including 219 HT,212 GD(121 untreated GD and 91 treated GD),19 subacute thyroiditis,20 nodular goiter and 14 simple goiter.The results were analyzed with SPSS 12.0 software.The differences among these groups were analyzed with t test,F test or x² test.Results:1.The recombinant prokaryotic expressive plasmid pET102/D-TOPO was extracted from E.coli TOP10,and purpose gene was identified by PCR.The yield of it reached 3.3 mg/L.The molecule weight of it was 72.68 kD,which is 2.66%above the theoretic evaluation(70.8 kD).Its immune activity was identified by ELISA.2.The maximum ratio of positive A₄₀₅to negative A₄₀₅was 3.291 that was above 2.1,when the definition of the optimum condition of ELISA was the antigen coating dose 1μg/well,the dilution of patients' sera 1:50,anti-Human IgG-Alkaline Phosphatase in Goat 1:20000,at 37℃,60 minutes.There was the significant correlation between ELISA and RIA(r=0.899,P＜0.01).The inter-assay coefficient of variation(CV)was 4.3%,4.7%(n=8)and the intra-assay CV was 4.7%,12.2%(n=7) for positive quality control sera and negative quality control sera,respectively.3.The results of clinical measurement using ELISA as follows:(1)In 300 healthy sera samples,there were neither significant differences among every age groups(F=0.655,P＞0.05),nor between men and women(t=1.68,P＞0.05).Therefore, these groups were put together as normal group.The value of TgAb in normal group was 0.404±0.173,the cut-off value was 0.74.(2)There was no difference between untreated GD and treated GD(t=0.690,P＞0.05).Therefore,these groups were put together as GD group.(3)The TgAb value in HT was much higher than GD (t=11.171,P＜0.01),nodular goiter(t=14.886,P＜0.01),simple goiter(t=15.984, P＜0.01),subacute thyroiditis(t=12.936,P＜0.01)or normal group(t=18.728,P＜0.01), respectively.The TgAb value in GD was much higher than nodular goiter(t=3.409, P＜0.01),simple goiter(t=3.606,P＜0.01)or normal group(t=4.775,P＜0.01), respectively.The TgAb value in subacute thyroiditis was higher than normal group (t=2.051,P＜0.05).There were no significant difference among other groups(P＞0.05). (4)The positive rates of HT,untreated GD,treated GD,subacute thyroiditis,nodular goiter and simple goiter were 70.8%,38.8%,28.6%,5.3%,5.0%and 0,respectively. The positive rate of combined GD group was 34.4%.The positive rate in HT was significant higher than that in other five group(P＜0.01,respectively).Even though there was no difference between in both GD,the positive rates in both GD were significant higher than those in nodular goiter,simple goiter or SAT(P＜0.01 or P＜0.05),respectively.There were not any differences of positive rates among nodular goiter,simple goiter or SAT(P＞0.05).Conclusions:1.The recombinant hTg can be expressed in prokaryotic system,and the product had immune activity.2.The ELISA to detect TgAb using recombinant hTg as antigen was established, and there was a significant correlation between ELISA and RIA method.3.784 specimens were measured using this ELISA.The results showed that it could be used in clinical application to identify thyroid diseases.