| Autoimmune thyroid disease(AITD)is a kind of disease including Graves disease(GD),Hashimoto's thyroiditis(HT)and idiopathic myxedema.GD is the most common type of thyrotoxicosis,which is a kind of organ specific increased secretion of thyroid hormone in autoimmune diseases.Thyrotropin receptor(TSHR)is the most important pathogen in pathological conditions which TSHR as antigen to stimulate the body to produce auto-antibody(TRAb).TRAb is the cause of GD and play an important role in the pathogenesis,development and prognosis of GD.TRAb belongs to heterogeneous polyclonal antibodies,which are divided into thyroid stimulating antibody(TSAb)and thyroid stimulating blocking antibody(TSBAb).TSAb combined with TSHR to stimulate follicular growth and thyroid hormone synthesis and secretion resulting in hyperthyroidism.TSBAb combined with TSHR could block the stimulatory effect of TSH on the thyroid,which can induce the occurrence of hypothyroidism.TSHR is the G protein coupled receptors,including extracellular domain,transmembrane region and the membrane area of three parts.TSAb binding sites are mainly concentrated in the TSHR ectodomain amino terminal and TSBAb binding targets mainly concentrated in the carboxyl terminal of TSHR extracellular domain.In our previous study,the recombinant thioredoxin-human thyrotropin receptor amino terminal fragment fusion protein(TrxFus-hTSHRn)and thioredoxin-human thyrotropin receptor carboxyl terminal fragment fusion protein(TrxFus-hTSHRc)were obtained,which are hot area of TSAb and TSBAb,respectively.Objective:To explore the fermentation technology of recombinant human TSHR extracellular domain in N-terminal and C-terminal antigen.Research and develope the TRAb(TSAb mainly)chemiluminescence enzyme immunoassay kit which is the TrxFus-hTSHRn(TSAb binding hot spots)as coated antigen and TRAb(TSBAb mainly)chemiluminescenceenzymeimmunoassaykitwhichis the TrxFus-hTSHRc(TSBAb binding hot spots)as coated antigen.Evaluate the clinical value of these two kits for AITD diagnosis,differential diagnosis,observation of curative effect and prognosis.Methods:1.The exploration of recombinant human TSHR ectodomain amino terminal and carboxyl terminalantigen fermentation processThe recombinant expression plasmid(pET102/D-hTSHRn462bp and pET102/D-hTSHRc405bp)were transferred into E.ColiBL21(DE3).Exploration on the optimization of 5 liter fermentor pilot fermentation conditions:fermentation temperature,pH,dissolved oxygen,inducer concentration,induction time.The expression products were purified by affinity chromatography and gradient dialysis refolding.The purified protein was identified the molecular weight and purity by SDS-PAGE,the immune activity was identified by Western blotting.2.The development of serum TRAb chemiluminescence enzyme immunoassay kitsDevelope the TRAb(TSAb mainly)chemiluminescence enzyme immunoassay kit which is the TrxFus-hTSHRn(TSAb binding hot spots)as coated antigen and TRAb(TSBAb mainly)chemiluminescence enzyme immunoassay kit which is the TrxFus-hTSHRc(TSBAb binding hot spots)as coated antigen.Optimization of experimental conditions:(1)antigen coated buffer(2)The amount of coating antigen and the dilution of serum to be tested(3)the reaction time.Methodological evaluation(1)the limit of detection(2)specificity(3)precision(4)accuracy(5)integrity(6)reference interval3.Clinical application of human serum TRAb chemiluminescence enzyme immunoassay kitsIn 597 cases of patients with thyroid disease and 120 healthy people were detected by TRAb/TSAb-CLEIA Kit and 416 cases of patients with thyroid disease and 120 healthy people were detected by TRAb/TSBAb-CLEIA Kit.Results:1.The exploration of recombinant human TSHR ectodomain amino terminal and carboxyl terminalantigen fermentation processTheoptimal fermentation conditionsofthe TrxFus-hTSHRn were:1mmol/L IPTG,6 hours of induction at 30?,culture pH 7,dissolved oxygen is greater than 10%.The molecular weight of TrxFus-hTSHRn is about 38KD,the purity of95.8%,and good immune activity binding with human serum TSAb,the yield of fermentation was 52.3-61.8mg/L medium.Theoptimal fermentation conditionsofthe TrxFus-hTSHRcwere:1mmol/L IPTG,6 hours of induction at 30?,culture pH 7,dissolved oxygen is greater than 20%.The molecular weight of TrxFus-hTSHRc is about 28.9KD,the purity of97.5%,and good immune activity binding with human serum TSBAb,the yield of fermentation was 67.3-81.6 mg/L medium.2.The development of serum TRAb chemiluminescence enzyme immunoassay kitsMethodological evaluation of TRAb/TSAb-CLEIA Kit:(1)the minimum detection limit:0.06 IU/L.(2)Specificity:cross reaction with TgAb and TPOAb were both less than 0.5%.(3)Precision:the intra-assay coefficient of variation(CV%)were5.0%,4.8%,5.7%(n=12).The inter-assay coefficient of variation(CV%)were4.7%,5.5%,5.4%(n=12).(4)Accuracy:the recovery of samples of different concentrations were 95.3%,106.3%,91.6%.(5)Integrity:the ratio of theoretical concentration and measured concentration after dilution in the standard range of 90%-110%.(6)The reference interval:serum TSAb concentration was less than 4IU/L as negative,more than 4 IU/L as positive.Methodological evaluation of TRAb/TSBAb-CLEIA Kit:(1)the minimum detection limit:0.15AU/L.(2)Specificity:cross reaction with TgAb and TPOAb were both less than4%.(3)Precision:the intra-assay coefficient of variation(CV%)were5.02%,4.67%,7.05%(n=12).The inter-assay coefficient of variation(CV%)were5.34%,2.82%,4.07%(n=12).(4)Accuracy:the recovery of samples of different concentrations were 96.8%,98.0%,104.0%.(5)Integrity:the ratio of theoretical concentration and measured concentration after dilution in the standard range of 90%-110%.(6)The reference interval:serum TSBAb concentration was less than 14 AU/L as negative,more than 14 AU/L as positive.3.Clinical application of human serum TRAb chemiluminescence enzyme immunoassay kits(1)The concentration of serum TSAb and the positive rate of GD in hyperthyroidism group was significantly higher than that of the GD treatment group,HT hypothyroidism group,nodular goiter,subacute thyroiditis group and the healthy control group.(2)Serum TSAb concentration in patients with GD during the course of treatment was significantly decreased,but still higher than that in healthy control group(F=3.272,P=0.002).At the same time,the positive rate of TSAb in patients with GD was significantly decreased,but still higher than that in healthy people.(3)The concentration of serum TSAb and the positive rate of in in nodular goiter,subacute thyroiditis patients and healthy controls showed no significant difference.(4)The concentration and the positive rate of of serum TSAb in HT patients with hypothyroidism were significantly higher than those of nodular goiter group,subacute thyroiditis group,healthy group.(5)The concentration of TSBAb and the positive rate of in HT with hypothyroidism group,in HT of normal thyroid function group were significantly higher than that in other groups(F=7.366,P=0.001;2=43.75,P=0.008).There was no significant difference of serum TSBAb concentrations in GDwith hyperthyroidismgroup,nodulargoiter,subacute thyroiditis group and healthy control group(F=2.39,P=0.12),but the positive rate in GD hyperthyroidism group was higher than that of the above three groups(?~2=27.11,P=0.03).Conclusions:1.The recombinant fusion protein TrxFus-hTSHRn and TrxFus-hTSHRc are of high purity(95.8%and 97.5%),good immune activity and high yield of fermentation(52.3-61.8mg/L medium and 67.3-81.6mg/L medium).2.TRAb/TSAb-CLEIA kit for quantitative detection of human serum TRAb(TSAb mainly)and TRAb/TSBAb-CLEIA kit for quantitative detection of human serum TRAb(TSBAb mainly).The kits have the advantages of high sensitivity,good specificity,good stability,simple operation,suitable for clinical routine examination.3.TSAb can be used as one of the best immunological indexes for diagnosis in GD hyperthyroidism,judgement of withdrawal time and prediction of recurrence. |
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