| Background and objectivesMost of Thyroxine (T4) and Triiodothyronine (T3) in serum are bound with thyroxine-binding globulin (TBG), Thyroxine-binding prealbumin(TBPA) and alblumn. About0.03%of T4and0.3%of T3, which are not attached to serum proteins but in free form (FT4, FT3), has been demonstrated to be the metabolically active fraction and a better barometer for the thyroid status of the host.The gold reference method that accurately detects serum FT4and FT3is equilibrium dialysis, but due to its high technical requirements, and the method is very time-consuming (usually16-24hours), thus limiting its widely use in the clinical laboratory. The rapid development of labelled immunoassay makes fast and accurate determination of FT4and FT3possible, and detection reagents for FT4and FT3using radioimmunoassay (RIA), chemilinescent Immunoassay (CLIA), time-resolved Fluoroimmunoassay (TRFIA) and electrical chemilinescent immunoassay (ECLIA) have emerged. These methods can be further divided into three categories:two-step method, one-step labelled analog method and labelled-antibody method. Although the accuracy of the two-step method is higher, the operation is complicated and time-consuming. The one-step labelled analog method is simple and has good precision, but its accuracy has been questioned. The labeled-antibody method that combines the respective advantages of the two-step method and the one-step labelled analog method, therefore is widely used by many commercial reagent company. No matter which method is used, the key difficulty lies in diminishing the destruction of equilibrium between the T4or T3and the binding protein during the determination process.Time-resolved fluoroimmunoassay (TRFIA) is a highly sensitive and quantitative immunoassay technology using antigens or antibodies labelled by rare earth ion. Due to the unique fluorescence properties of lanthanide rare earth ions, the technique has advantages of high signal to noise ratio, hence has high sensitivity, simple labelling process, long storage time, no radioactive contamination, good repeatability, simple operation process, wide range of detection, not sensitive to natural fluorescence interference and very wide range of applications.TRFIA was used in this thesis for the deep and extensive research on FT4and FT3determination of human serum. We established a rapid, sensitive, reliable FT4/FT3TRFIA by labelled-antibody method.The thesis consists of four parts, which are review, synthesis of five thyroid hormone derivatives and preparation of conjugates, the TRFIA research on serum FT4and FT3, respectively.The review focuses on the methodology characteristics time-resolved fluorescence immunoassay and the progress of detection method of FT4and FT3.In the second part we described the synthesis of five types of thyroid hormone derivatives and preparation of the corresponding conjugates in detail, aiming to provide an important foundation of raw materials for the third and fourth part of the methodological research;In the third and the fourth part of the thesis we investigated the measurment of serum FT4and FT3based on TRFIA, respectively. Firstly, we determined the optimal conjugate comparing different sensitivities obtained by different conjugates, and then established the FT4/FT3TRFIA based on the optimal conjugate, and investigated the detailed performance evaluation and identification.Methods(一) Five thyroid derivatives were synthesized by chemical synthesis and the corresponding rabbit IgG conjugates were prepared at the same time.1. The bis-NHS ester structure of the suberic acid was synthesized with the interactions of DCC and NHS using suberic acid as an initial material.2. T4and T3were used as synthetic starting materials to obtain methyl T4and T3methyl ester in a methanol solution of HCl, respectively.3. T4, T3, T4methyl ester, T3methyl ester, and T2were used as the starting materials, reacting with the bis-NHS ester of suberic acid with a molar ratio1:2.1to form the NHS ester structures of derivatives, respectively.4. The mixture of the five derivatives NHS ester with rabbit IgG (molar ratio of100:1) were stirred at room temperature for20h respectively, and the final products were purified by molecular sieve and the concentrations of the five conjugates were determined using the BCA method, respectively.(二) The establishment of serum FT4time-resolved fluoroimmunoassay1. The hormone-free human serum was obtained by treatment of activated charcoal and kaolin,which was used as the matrix of calibrators.2. FT4calibrators were prepared using the pure T4antigen and the hormone-free human serum, and calibrated using the commercialized DELFIA FT4assay. The concentrations of six FT4calibrators were0pmol/L,2.5pmol/L,6.6pmol/L,15pmol/L,42pmol/L and120pmol/L, respectively.3. Preparation of Eu3+-labeled anti-T4monoclonal antibody:the pretreated antibody was added to DTTA-Eu3+labeled reagent and were mixed thoroughly and allowed to stand at room temperature overnight. The resulting product were subjected to molecular sieve for purification the next day. The labeled antibody were collected and filtered through0.22μm microporous filter, the concentration of the labeled antibody was determined using the BCA protein assay kit, and finally adding europium-labeled protein stabilizer to a final concentration of0.2%.4. Preparation of the96-well coated plate:a known concentration of conjugate was diluted to a final concentration using coating buffer,150μl was added to each well, and allowed to stand for20hours at37℃incubator. After washing3times and patting dry,280μl blocking solution was added to each well, then the plate was allowed to stand for12hours at4℃. The plate was patted dry.5. Detection of FT4using labeled antibody TRFIA25uL calibrators or serum samples together with150μL of the diluted labelled anti-T4monoclonal antibody in assay buffer were added in duplicates in the wells. After incubation with shaking at37℃for60min, the microstrips were washed six times and patted dry.150μl enhancement solution was added to each well. The plate was incubated at room temperature for five minutes with gentle shaking. The fluorescence in each well was measured with a DELFIA1235analyser.6. Determination of the optimal conjugateT4-IgG, T4methyl ester-IgG and T3-IgG were coated at the final concentration of2μg/ml, respectively, then the FT4TRFIA analysis was in progress and FT4standard curve was plotted. The optimal conjugate was chosen based on the the ED50and Bmax. 7. Assay optimizationThe optimal coating concentration, the optimal dilution of labelled antibody and the optimal incubation time were determined.8. Evaluating the performance of TRFIA for the FT4detection8.1Calibration curve and determination of analytical sensitivityCalibration curve was plotted using the following parameters:X=log(concentration), Y=Logit (B/Bmax)=In ((B/Bmax)/(1-B/Bmax)), and the correlation coefficient was obtained. The fluorescence of zero calibrator was parallel determined ten times and standard deviation(SD) was calculated, and analytical sensitivity was calculated by analytical software as the concentration of FT4that corresponded to the fluorescence counts that were two standard deviations less than the mean fluorescence counts of10replicate determinations of the FT4zero standard.8.2Precision assayPrecision was evaluated using three different FT4sample concentrations, each sample was parallel determined ten times. The coefficient of variation (CV), including intra-assay and inter-assay, was calculated respectively.8.3Specificity assay(一)The specificity was investigated by adding T3, reverse T3and T2, respectively. the cross-reacting substance concentration at50%inhibition of maximum binding was obtained.(二)The concentration of T4at50%inhibition of maximum binding in total T4assay. Cross-reactions is defined as:the ratio of the latter to the former x100%.8.4Anti-interference analysis of thyroid-related binding proteinsVarious amount of thyroxine-binding proteins (TBG), human serum albumin (HSA) or prealbumin were added to the zero standard repectively. Different final concentrations of TBG (20,40,60and100mg/L), HSA (20,40,60and100g/L) and Prealbumin (100,200,300and500mg/L) in the zero standard were prepared. The changes of fluorescence counts between the zero standard without added protein and the one with added protein were analyzed.8.5Interference analysis of free fatty acidsDifferent concentration of1.1ml oleic acid were added to the lyophilized standard of DELFIA FT3kit marked as D (The concentration in this kit was marked as15.2pmol/L) respectively, dissolved fully and finally gave four different concentration of oleic acid sample(1,2,3, and5mmol/L). The lyophilized calibrator marked as D was dissolved with1.1ml purified water and was used as a control (calculated as100%), calculating the changes of FT4concentration after adding the oleic acid.8.6Comparison with imported DELFIA FT4AssayThe imported PerkinElmer DELFIA FT4kit was used as control to evaluate the effectiveness of our established FT4TRFIA.110serum samples were tested using two methods at the same time, and the correlation coefficient were calculated.(三)The establishment of serum FT3time-resolved fluoroimmunoassay1. The hormone-free human serum was obtained by treatment of activated charcoal and kaolin, which was used as the matrix of calibrators.2. FT3calibrators were prepared using the pure T3antigen and the hormone-free human serum, and calibrated using the commercialized DELFIA FT3assay. The concentrations of six FT3calibrators were0pmol/L,2.0pmol/L,4.0pmol/L,8.0pmol/L,16pmol/L and32pmol/L, respectively.3. Preparation of Eu3+-labeled anti-T3polyclonal antibody:the pretreated antibody was added to DTTA-Eu3+l abeled reagent and were mixed thoroughly and allowed to stand at room temperature overnight. The resulting product were subjected to molecular sieve for purification the next day. The labeled antibody were collected and filtered through0.22μm microporous filter, the concentration of the labeled antibody was determined using the BCA protein assay kit, and finally adding europium-labeled protein stabilizer to a final concentration of0.2%.4. Preparation of the96-well coated plate:a known concentration of conjugate was diluted to a final concentration using coating buffer,150μl was added to each well, and allowed to stand for20hours at37℃incubator. After washing3times and patting dry,280μl blocking solution was added to each well, then the plate was allowed to stand for12hours at4℃. The plate was patted dry.5. Detection of FT3using labeled antibody TRFIA25μL calibrators or serum samples together with150μL of the diluted labelled anti-T4monoclonal antibody in assay buffer were added in duplicates in the wells. After incubation with shaking at37℃for60min, the microstrips were washed six times and patted dry.150μl enhancement solution was added to each well. The plate was incubated at room temperature for five minutes with gentle shaking. The fluorescence in each well was measured with a DELFIA1235analyser.6. Determination of the optimal conjugateT2-IgG, T3methyl ester-IgG and T3-IgG were coated at the final concentration of2μg/ml, respectively, then the FT3TRFIA analysis was in progress and FT3standard curve was plotted. The optimal conjugate was chosen based on the the ED50and Bmax.7. Assay optimizationThe optimal coating concentration, the optimal dilution of labelled antibody and the optimal incubation time were determined.8. Evaluating the performance of TRFIA for the FT3detection8.1Calibration curve and determination of analytical sensitivity Calibration curve was plotted using the following parameters:X=log(concentration), Y=Logit (B/Bmax)=In ((B/Bmax)/(1-B/Bmax)), and the correlation coefficient was obtained. The fluorescence of zero calibrator was parallel determined ten times and standard deviation(SD) was calculated, and analytical sensitivity was calculated by analytical software as the concentration of FT3that corresponded to the fluorescence counts that were two standard deviations less than the mean fluorescence counts of10replicate determinations of the FT3zero standard.8.2Precision assayPrecision was evaluated using three different FT3sample concentrations, each sample was parallel determined ten times. The coefficient of variation (CV), including intra-assay and inter-assay, was calculated respectively.8.3Specificity assay(一)The specificity was investigated by adding T4, reverse T3and T2, respectively. the cross-reacting substance concentration at50%inhibition of maximum binding was obtained.(二)The concentration of T3at50%inhibition of maximum binding in total T3assay.Cross-reactions is defined as:the ratio of the latter to the former x100%.8.4Anti-interference analysis of thyroid-related binding proteinsVarious amount of thyroxine-binding proteins (TBG), human serum albumin (HSA) or prealbumin were added to the zero standard repectively. Different final concentrations of TBG (20,40,60and100mg/L), HSA (20,40,60and100g/L) and Prealbumin (100,200,300and500mg/L) in the zero standard were prepared. The changes of fluorescence counts between the zero standard without added protein and the one with added protein were analyzed.8.5Interference analysis of free fatty acids Different concentration of1.1ml oleic acid were added to the lyophilized standard of DELFIA FT3kit marked as D (The concentration in this kit was marked as9.4pmol/L) respectively, dissolved fully and finally gave four different concentration of oleic acid sample(1,2,3, and5mmol/L). The lyophilized calibrator marked as D was dissolved with1.1ml purified water and was used as a control (calculated as100%), calculating the changes of FT4concentration after adding the oleic acid.8.6Comparison with imported DELFIA FT3AssayThe imported PerkinElmer DELFIA FT3kit was used as control to evaluate the effectiveness of our established FT3TRFIA.110serum samples were tested using two methods at the same time, and the correlation coefficient were calculated.Results(一)The synthesis of five thyroid hormones derivatives and the preparation of corresponding rabbit IgG conjugatesWe synthesized five thyroid hormone derivatives successfully, and prepared the five rabbit IgG conjugates by coupling with the five thyroid hormones derivatives, respectively. The concentrations of T4-IgG, T3-IgG, T4methyl ester-IgG, T3methyl ester-IgG and T2-IgG were1.0mg/ml,1.1mg/ml,0.9mg/ml,1.0mg/ml and1.1mg/ml, respectively.(二)The developed serum FT4TRFIA1. The concentration of Eu3+labeled anti-T4monoclonal antibody is determined as130μg/ml by BCA protein assay;2. Comparing three conjugate based on Bmax and ED50, including T4-IgG, T4methyl ester-IgG and T3-IgG, we selected T3-IgG conjugate as the optimal coating material;3. Amount of the coating, the dilution of the labeled antibody, and the reaction time was optimized. The optimal coating amount was3μg/ml, the optimal dilution of the labeled antibody was1:1000and the optimum reaction time was1h;4. The FT4standard curve equation:Y=-1.146X+1.324, correlation r=0.9997, and the calculated analytical sensitivity was0.2pmol/L;5. PrecisionThe intra-assay coefficient of variation was3.5-6.6%and the inter-assay coefficient of variation was4.4-9.8%, bearing good precision.6. SpecificityCross-reaction with the T3is1.5%, and with reverse T3is1.2%, and with T2is less than0.1%.7. Anti-interference analysis of thyroid hormone-related binding proteinsThe established FT4TRFIA is not sensitive to the concentration changes of TBG, HSA and Prealbumin, basically reflecting no interference.8. Interference of free fatty acidWhen oleic acid reached2mmol/L, basically no interference on the proposed FT4TRFIA was observed, but when the concentration of oleic acid rose to5mmol/L, increased the estimate of FT4by15%, suggesting potential interference of oleic acid.9. Comparison with DELFIA FT4AssayBoth methods detected110samples, Good agreements between the two FT4methods were achieved with the square of the correlation of0.984, suggesting the method can meet the requirements of clinical testing.(三)The developed serum FT3TRFIA1. The concentration of Eu3+labeled anti-T3polyclonal antibody is determined as150ug/ml by BCA protein assay;2. Comparing three conjugate based on Bmax and ED50, including T2-IgG, T3methyl ester-IgG and T3-IgG, we selected T2-IgG conjugate as the optimal coating material; 3. Amount of the coating, the dilution of the labeled antibody, and the reaction time was optimized. The optimal coating amount was2.5μg/ml, the optimal dilution of the labeled antibody was1:800and the optimum reaction time was1h;4. The FT3standard curve equation:Y=-2.344X+1.98, correlation r=0.9998, and the calculated analytical sensitivity was0.3pmol/L;5. PrecisionThe intra-assay coefficient of variation was4.4-6.8%and the inter-assay coefficient of variation was4.8-9.6%, bearing good precision.6. SpecificityCross-reaction with the T4is0.2%, and with reverse T3is less than0.1%, and with T2is0.4%.7. Anti-interference analysis of thyroid hormone-related binding proteinsThe established FT3TRFIA is not sensitive to the concentration changes of TBG, HSA and Prealbumin, basically reflecting no interference.8. Interference of free fatty acidWhen oleic acid reached2mmol/L, basically no interference on the proposed FT3TRFIA was observed, but when the concentration of oleic acid rose to5mmol/L, increased the estimate of FT4by10%, suggesting potential interference of oleic acid.9. Comparison with DELFIA FT3AssayBoth methods detected110samples, Good agreements between the two FT4methods were achieved with the square of the correlation of0.966, suggesting the method can meet the requirements of clinical testing.ConclusionsThese results demonstrated that the proposed FT4/FT3TRFIA had good performance, including accuracy, sensitivity, precision, specificity, anti-interference performance, which could meet the requirements of clinical application. So these reagents are expected to replace and compete with expensive imported kits. |
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