游离甲状腺素与游离三碘甲状腺原氨酸的时间分辨荧光免疫检测试剂的研究
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摘要
研究背景及目的
人体血清中的甲状腺素(Thyroxine, T4)和三碘甲状腺原氨酸(Triiodothyronine, T3)绝大部分是与甲状腺结合球蛋白(thyroxine-binding globulin, TBG)、甲状腺结合前白蛋白(Thyroxine-binding prealbumin, TBPA)和白蛋白结合,仅有约0.03%的T4和0.3%的T3以游离的状态存在。然而,正是这些极微量的游离分子(FT4, FT3)发挥着人体代谢的生理作用,成为评价人体甲状腺功能的重要指标。
平衡透析法是准确测定人血清中FT4和FT3的金标准方法,但由于其技术要求较高而且操作相当费时(通常需要16-24小时),因而限制了其在临床实验室的广泛使用。标记免疫学的迅猛发展使得FT4和FT3的快速准确测定成为可能,现今已经出现了如放射免疫分析(Radioimmunoassay, RIA)、化学发光免疫分析(Chemilinescent Immunoassay, CLIA)、时间分辨荧光免疫分析(Time-resolved Fluoroimmunoassay, TRFIA)及电化学发光(Electrical Chemilinescent Immunoassay, ECLIA)等这些方法学的FT4/FT3检测试剂。将这些方法学进一步分类,可将FT4的检测试剂分为三类：两步法、一步类似物法和标记抗体法。两步法尽管测值准确性较高,但操作复杂,耗时长；一步类似物法操作简便,精密度好,但准确性一直备受质疑；标记抗体法是目前许多商业化试剂公司普遍采用的方法,它综合了两步法和一步类似物法各自的优点,因此被越来越广泛的运用。实际上,无论采用哪种方法,其中的关键难点在于测定的过程中尽量最少程度的破坏T4或T3与结合蛋白之间的平衡。
时间分辨荧光免疫分析(Time-resolved fluoroimmunoassay, TRFIA)是一种高灵敏的定量免疫检测技术,其独特的地方在于用镧系稀土离子作为标记物来标记抗原或抗体,因镧系稀土离子具有独特的荧光特性,该技术能够获得极高的信噪比,从而具有很高的灵敏度,且还具有标记物制备简便、储存时间长、无放射性污染、检测重复性好、操作流程短、标准曲线范围宽、不受样品自然荧光干扰和应用范围广泛等优点。
本论文采用时间分辨荧光免疫分析法对人血清中的FT4与FT3检测进行了深入广泛的研究。我们采用标记抗体法建立了一套快速、灵敏、可靠的FT4/FT3时间分辨荧光免疫分析方法。
本论文分为四个部分,分别为综述、五种甲状腺激素衍生物的合成以及偶联物的制备、血清FT4的TRFIA研究、血清FT3的TRFIA研究。
论文的综述部分详细介绍了时间分辨荧光免疫分析法的方法学特点和FT4/FT3检测方法的进展情况；
论文的第二部分详细描述了五种甲状腺激素衍生物的合成以及偶联物的制备情况,旨在为论文的第三、四部分的方法学研究提供重要的原料基础；
论文的第三部分与第四部分以Eu3+为标记物分别对血清FT4和FT3的TRFIA进行了研究,我们首先通过比较不同的偶联物产生的灵敏度高低差异来选取灵敏度最高的确定为最佳的偶联物,然后用此最佳偶联物来建立FT4/FT3的TRFIA,并进行了详细的性能评价和鉴定。
方法：
(一)采用化学合成的方法合成了五种甲状腺激素衍生物并分别将其与兔IgG偶联制备了其相应的兔IgG偶联物。
1.以辛二酸作为起始原料,在DCC和NHS的作用下反应得到辛二酸的双NHS酯结构；
2.以T4、T3作为合成起始原料在HCL的甲醇溶液中反应分别得到T4甲酯和T3甲酯；
3.以T4、T3、T4甲酯、T3甲酯和T2作为合成起始原料与辛二酸的NHS酯以摩尔比为1：2.1反应形成各自的衍生物的单NHS酯结构；
4.将五种衍生物的单NHS酯与兔IgG以摩尔比100：1的比例进行偶联反应,反应于室温下搅拌20h后用分子筛进行纯化,并用BCA法分别测定这五种偶联物浓度。
(二)血清FT4时间分辨荧光免疫分析法的建立
1.利用活性炭、高岭土制备去激素人血清作为校准品配制用基质。
2.采用T4抗原配制FT4校准品,并用商业化的DELFIA FT4assay进行标定,标定后的校准品浓度依次为：0pmol/L,2.5pmol/L,6.6pmol/L,15pmol/L,42pmol/L,120pmol/L。
3.Eu3+标记抗T4单克隆抗体的制备：抗体经预处理后和标记试剂DTTA-Eu3+充分混匀,置于室温过夜,次日上样过分子筛纯化。收集纯化后的目的产物,经0.22μm微孔过滤器过滤,用BCA蛋白定量试剂盒对标记抗体的浓度进行定量,最后加入铕标稳定液至终浓度为0.2%。
4.96孔包被反应板的制备：将已知浓度的偶联物用包被液稀释成某一终浓度,每孔加入150μl，37℃恒温箱中静置20小时。洗板3次后拍干,加入封闭液280μl/孔,4℃静置12h。直接倒去封闭液,拍干。
5. TRFIA标记抗体法检测FT4
每孔加入25μl血清样本或校准品,将标记抗体用分析缓冲液以一定比例稀释,每孔加入稀释后的标记抗体150μl。37℃振荡孵育1h,洗涤6次,拍干,加150μl增强液,室温慢速振荡孵育5分钟。然后在PerkinElmer1235分析仪上测定荧光值。
6.最佳的偶联物的确定
分别包被T4-IgG、T4甲酯-IgG和T3-IgG三种偶联物,其包被终浓度均为2μg/ml,进行FT4的TRFIA分析,得到FT4的标准曲线。依据ED50和Bmax来选取最佳偶联物。
7.系统优化：对最佳包被量、标记抗体的最佳稀释度以及最佳反应时间进行确定。
8.自制的TRFIA FT4检测方法的性能评估
8.1标准曲线和分析灵敏度的确定
以X=Ln(浓度),Y=Logit (B/Bmax)=Ln ((B/Bmax)/(1-B/Bmax)),以此绘制标准曲线,计算线性相关性。平行测定十次校准品A的荧光值,并计算其标准差,用A点荧光值的平均值减去2倍标准差得到的荧光值,依据PerkinElmer1235分析仪自带的软件可计算出所对应的浓度,此浓度即为分析灵敏度。
8.2精密度
对低、中、高三个不同浓度的FT4样本进行重复测定10次,计算分析内和分析间变异系数(CV)。
8.3特异性
(一)计算加入的待测交叉反应物质T3,反T3及T2分别能达到50%抑制时的浓度；(二)总T4分析本身达到50%抑制时T4的浓度。
交叉反应定义为：后者与前者的比值×100%。
8.4甲状腺激素相关结合蛋白的抗干扰分析
将一定量的TBG、HSA和Prealbumin加至零点校准品中。最终得到分别含有4个不同终浓度的TBG (20,40,60和100mg/L), HSA (20,40,60和100g/L)and Prealbumin (100,200,300和500mg/L)零点校准品。比较未添加和添加相关蛋白的零点标准品荧光值的变化。
8.5游离脂肪酸的抗干扰分析
将1.1ml不同浓度的水相油酸分别加至PerkinElmer DELFIA FT4试剂盒中的D点冻干校准品中(该试剂盒标示的浓度为15.2pmol/L),充分溶解,最终得到4个含有不同浓度(1,2,3和5mmol/L)的油酸的校准品。以加入1.1ml纯化水溶解的D点冻干校准品的浓度作为对照(当作100%),比较添加油酸后的校准品测定的FT4的浓度值变化。
8.6与进口试剂的相关性比较
用进口PerkinElmer DELFIA FT4试剂盒作为对照方法以评价我们建立的FT4的TRFIA的有效性。110份血清样本同时用两种方法进行检测,计算其相关系数。
(三)血清FT3时间分辨荧光免疫分析法的建立
1.利用活性炭、高岭土制备去激素人血清作为校准品配制用基质。
2.采用T3抗原配制FT3校准品,并用商业化的DELFIA FT3assay进行标定,标定后的校准品浓度依次为：0pmol/L,2.0pmol/L,4.0pmol/L,8.0pmol/L,16pmol/L,32pmol/L。
3.Eu3+标记抗T3多克隆抗体的制备：抗体经预处理后和标记试剂DTTA-Eu3+充分混匀,置于室温过夜,次日上样过分子筛纯化。收集纯化后的目的产物,经0.22μm微孔过滤器过滤,用BCA蛋白定量试剂盒对标记抗体的浓度进行定量,最后加入铕标稳定液至终浓度为0.2%。
4.96孔包被反应板的制备：将已知浓度的偶联物用包被液稀释成某一终浓度,每孔加入150μl,37℃恒温箱中静置20小时。洗板3次后拍干,加入封闭液280μl/孔,4℃静置12h。直接倒去封闭液,拍干。
5. TRFIA标记抗体法检测FT3
每孔加入25μl血清样本或校准品,将标记抗体用分析缓冲液以一定比例稀释,每孔加入稀释后的标记抗体150μl。37℃振荡孵育1h,洗涤6次,拍干,加150μl增强液,室温慢速振荡孵育5分钟。然后在PerkinElmer1235分析仪上测定荧光值。
6.最佳的偶联物的确定
分别包被T2-IgG、T3甲酯-IgG和T3-IgG三种偶联物,其终浓度均为2μg/ml,进行FT3的TRFIA分析,得到FT3标准曲线。依据ED50和Bmax来选取最佳偶联物。
7.系统优化：对最佳包被量、标记抗体的最佳稀释度以及最佳反应时间进行确定。
8.自制的TRFIA FT3检测方法的性能评估
8.1标准曲线和分析灵敏度的确定
以X=Ln(浓度),Y=Logit (B/Bmax)=Ln ((B/Bmax)/(1-B/Bmax),以此绘制标准曲线,计算线性相关性。平行测定十次校准品A的荧光值,并计算其标准差,用A点荧光值的平均值减去2倍标准差得到的荧光值,依据PerkinElmer1235分析仪自带的软件可计算出所对应的浓度,此浓度即为分析灵敏度。
8.2精密度
对低、中、高三个不同浓度的FT3样本进行重复测定10次,计算分析内和分析间变异系数(CV)。
8.3特异性
(一)计算加入的待测交叉反应物质T4,反T3及T2分别能达到50%抑制时的浓度；(二)总T3分析本身达到50%抑制时T3的浓度。
交叉反应定义为：后者与前者的比值×100%。
8.4甲状腺激素相关结合蛋白的抗干扰分析
将一定量的TBG、HSA和Prealbumin加至零点校准品中。最终得到分别含有4个不同终浓度的TBG (20,40,60和100mg/L), HSA (20,40,60和100g/L)and Prealbumin (100,200,300和500mg/L)零点校准品。比较未添加和添加相关蛋白的零点标准品荧光值的变化。
8.5游离脂肪酸的抗干扰分析
将1.1ml不同浓度的水相油酸分别加至PerkinElmer DELFIA FT3试剂盒中的D点冻干校准品中(该试剂盒标示的浓度为9.4pmol/L),充分溶解,最终得到4个含有不同浓度(1,2,3和5mmol/L)的油酸的校准品。以加入1.1ml纯化水溶解的D点冻干校准品的浓度作为对照(当作100%),比较添加油酸后的校准品测定的FT3的浓度值变化。
8.6与进口试剂的相关性比较
用进口PerkinElmer DELFIA FT3试剂盒作为对照方法以评价我们建立的FT3的TRFIA的有效性。110份血清样本同时用两种方法进行检测,计算其相关系数。
结果：
(一)五种甲状腺激素的衍生物的合成和与兔IgG偶联物的制备
成功合成了五种甲状腺激素的衍生物,并分别与兔IgG发生偶联得到五种偶联物T4-IgG、T3-IgG、T4甲酯-IgG、T3甲酯-IgG和T2-IgG。其浓度分别为：1.0mg/ml、1.1mg/ml、0.9mg/ml、1.0mg/ml和1.1mg/ml。
(二)血清FT4时间分辨荧光免疫分析法的建立
1.制备的Eu3+标记抗T4单克隆抗体的浓度为130μg/ml;
2.通过比较T4-IgG、T4甲酯-IgG和T3-IgG三种偶联物产生的灵敏度高低,我们选取了T3-IgG作为最佳的包被偶联物；
3.对包被量、标记抗体的稀释度以及反应时间进行优化分析,得出：最佳的包被量为3μg/ml;标记抗体的最佳稀释度为1：1000；最佳反应时间为1h；
4.FT4的标准曲线方程为：Y=-1.146X+1.324,相关性r=0.9997,计算得到的分析灵敏度为0.2pmol/L;
5.精密度
分析内变异系数为3.5-6.6%,分析间变异系数为和4.4-9.8%,精密度良好。
6.特异性
与T3的交叉反应率为1.5%,与反T3的交叉反应率为1.2%,与T2的交叉反应率小于0.1%。
7.甲状腺激素相关结合蛋白的抗干扰分析
建立的FT4TRFIA对于三种结合蛋白TBG、HSA以及Prealbumin的浓度变化不敏感,基本没有干扰。
8.游离脂肪酸的抗干扰分析
当样本中油酸的浓度为2mmol/L时,基本对本检测方法不会有干扰,但当样本中油酸的浓度升至5mmol/L时,会使FT4的测值偏高15%左右。
9.与PE进口试剂的相关性比较
两种方法均检测110例样本,本研究方法与对照PE试剂盒相关性达到R2=0.984,能够很好的满足临床检测的要求。
(三)血清FT3时间分辨荧光免疫分析法的建立
1.制备的Eu3+标记抗T3单克隆抗体的浓度为150μg/ml；
2.通过比较T2-IgG.T3甲酯-IgG和T3-IgG三种偶联物产生的灵敏度高低,我们选取了T2-IgG作为最佳的包被偶联物；
3.对包被量、标记抗体的稀释度以及反应时间进行优化分析,得出：最佳的包被量为2.5μg/ml;标记抗体的最佳稀释度为1：800；最佳反应时间为1h；
4.FT3的标准曲线方程为：Y=-2.344X+1.980,相关性r=0.9998,计算得到的分析灵敏度为0.3pmol/L；
5.精密度
分析内变异系数为4.4-6.8%,分析间变异系数为4.8-9.6%,精密度良好。
6.特异性
与T4的交叉反应率为0.2%,与反T3的交叉反应率小于0.1%,与T2的交叉反应率为0.4%。
7.甲状腺激素相关结合蛋白的抗干扰分析
建立的FT3TRFIA对于三种结合蛋白TBG.HSA以及Prealbumin的浓度变化不敏感,基本没有干扰。
8.游离脂肪酸的抗干扰分析
当样本中油酸的浓度为2mmol/L时,基本对本检测方法不会有干扰,但当样本中油酸的浓度升至5mmol/L时,会使FT3的测值偏高不到10%。
9.与进口试剂的相关性比较
两种方法均检测110例样本,本研究方法与对照PE试剂盒相关性达到R2=0.966,能够很好的满足临床检测的要求。
结论：
上述结果表明本论文建立的FT4和FT3时间分辨荧光免疫测定方法各项指标(准确性、灵敏度、精密性、特异性、抗干扰性等)均能够满足临床检测试剂要求,可与国外价格高昂的同类试剂相竞争。
Background and objectives
Most 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 fluoroimmunoassay
1. 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 TRFIA
25uL 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 conjugate
T4-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 optimization
The optimal coating concentration, the optimal dilution of labelled antibody and the optimal incubation time were determined.
8. Evaluating the performance of TRFIA for the FT4detection
8.1Calibration 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 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 assay
Precision 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 proteins
Various 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 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 FT4Assay
The 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 fluoroimmunoassay
1. 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 TRFIA
25μL 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 conjugate
T2-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 optimization
The optimal coating concentration, the optimal dilution of labelled antibody and the optimal incubation time were determined.
8. Evaluating the performance of TRFIA for the FT3detection
8.1Calibration 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 assay
Precision 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 proteins
Various 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 FT3Assay
The 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 conjugates
We 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 FT4TRFIA
1. 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. Precision
The intra-assay coefficient of variation was3.5-6.6%and the inter-assay coefficient of variation was4.4-9.8%, bearing good precision.
6. Specificity
Cross-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 proteins
The established FT4TRFIA is not sensitive to the concentration changes of TBG, HSA and Prealbumin, basically reflecting no interference.
8. Interference of free fatty acid
When 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 FT4Assay
Both 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 FT3TRFIA
1. 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. Precision
The intra-assay coefficient of variation was4.4-6.8%and the inter-assay coefficient of variation was4.8-9.6%, bearing good precision.
6. Specificity
Cross-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 proteins
The established FT3TRFIA is not sensitive to the concentration changes of TBG, HSA and Prealbumin, basically reflecting no interference.
8. Interference of free fatty acid
When 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 FT3Assay
Both 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.
Conclusions
These 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.
人体血清中的甲状腺素(Thyroxine, T4)和三碘甲状腺原氨酸(Triiodothyronine, T3)绝大部分是与甲状腺结合球蛋白(thyroxine-binding globulin, TBG)、甲状腺结合前白蛋白(Thyroxine-binding prealbumin, TBPA)和白蛋白结合,仅有约0.03%的T4和0.3%的T3以游离的状态存在。然而,正是这些极微量的游离分子(FT4, FT3)发挥着人体代谢的生理作用,成为评价人体甲状腺功能的重要指标。
平衡透析法是准确测定人血清中FT4和FT3的金标准方法,但由于其技术要求较高而且操作相当费时(通常需要16-24小时),因而限制了其在临床实验室的广泛使用。标记免疫学的迅猛发展使得FT4和FT3的快速准确测定成为可能,现今已经出现了如放射免疫分析(Radioimmunoassay, RIA)、化学发光免疫分析(Chemilinescent Immunoassay, CLIA)、时间分辨荧光免疫分析(Time-resolved Fluoroimmunoassay, TRFIA)及电化学发光(Electrical Chemilinescent Immunoassay, ECLIA)等这些方法学的FT4/FT3检测试剂。将这些方法学进一步分类,可将FT4的检测试剂分为三类：两步法、一步类似物法和标记抗体法。两步法尽管测值准确性较高,但操作复杂,耗时长；一步类似物法操作简便,精密度好,但准确性一直备受质疑；标记抗体法是目前许多商业化试剂公司普遍采用的方法,它综合了两步法和一步类似物法各自的优点,因此被越来越广泛的运用。实际上,无论采用哪种方法,其中的关键难点在于测定的过程中尽量最少程度的破坏T4或T3与结合蛋白之间的平衡。
时间分辨荧光免疫分析(Time-resolved fluoroimmunoassay, TRFIA)是一种高灵敏的定量免疫检测技术,其独特的地方在于用镧系稀土离子作为标记物来标记抗原或抗体,因镧系稀土离子具有独特的荧光特性,该技术能够获得极高的信噪比,从而具有很高的灵敏度,且还具有标记物制备简便、储存时间长、无放射性污染、检测重复性好、操作流程短、标准曲线范围宽、不受样品自然荧光干扰和应用范围广泛等优点。
本论文采用时间分辨荧光免疫分析法对人血清中的FT4与FT3检测进行了深入广泛的研究。我们采用标记抗体法建立了一套快速、灵敏、可靠的FT4/FT3时间分辨荧光免疫分析方法。
本论文分为四个部分,分别为综述、五种甲状腺激素衍生物的合成以及偶联物的制备、血清FT4的TRFIA研究、血清FT3的TRFIA研究。
论文的综述部分详细介绍了时间分辨荧光免疫分析法的方法学特点和FT4/FT3检测方法的进展情况；
论文的第二部分详细描述了五种甲状腺激素衍生物的合成以及偶联物的制备情况,旨在为论文的第三、四部分的方法学研究提供重要的原料基础；
论文的第三部分与第四部分以Eu3+为标记物分别对血清FT4和FT3的TRFIA进行了研究,我们首先通过比较不同的偶联物产生的灵敏度高低差异来选取灵敏度最高的确定为最佳的偶联物,然后用此最佳偶联物来建立FT4/FT3的TRFIA,并进行了详细的性能评价和鉴定。
方法：
(一)采用化学合成的方法合成了五种甲状腺激素衍生物并分别将其与兔IgG偶联制备了其相应的兔IgG偶联物。
1.以辛二酸作为起始原料,在DCC和NHS的作用下反应得到辛二酸的双NHS酯结构；
2.以T4、T3作为合成起始原料在HCL的甲醇溶液中反应分别得到T4甲酯和T3甲酯；
3.以T4、T3、T4甲酯、T3甲酯和T2作为合成起始原料与辛二酸的NHS酯以摩尔比为1：2.1反应形成各自的衍生物的单NHS酯结构；
4.将五种衍生物的单NHS酯与兔IgG以摩尔比100：1的比例进行偶联反应,反应于室温下搅拌20h后用分子筛进行纯化,并用BCA法分别测定这五种偶联物浓度。
(二)血清FT4时间分辨荧光免疫分析法的建立
1.利用活性炭、高岭土制备去激素人血清作为校准品配制用基质。
2.采用T4抗原配制FT4校准品,并用商业化的DELFIA FT4assay进行标定,标定后的校准品浓度依次为：0pmol/L,2.5pmol/L,6.6pmol/L,15pmol/L,42pmol/L,120pmol/L。
3.Eu3+标记抗T4单克隆抗体的制备：抗体经预处理后和标记试剂DTTA-Eu3+充分混匀,置于室温过夜,次日上样过分子筛纯化。收集纯化后的目的产物,经0.22μm微孔过滤器过滤,用BCA蛋白定量试剂盒对标记抗体的浓度进行定量,最后加入铕标稳定液至终浓度为0.2%。
4.96孔包被反应板的制备：将已知浓度的偶联物用包被液稀释成某一终浓度,每孔加入150μl，37℃恒温箱中静置20小时。洗板3次后拍干,加入封闭液280μl/孔,4℃静置12h。直接倒去封闭液,拍干。
5. TRFIA标记抗体法检测FT4
每孔加入25μl血清样本或校准品,将标记抗体用分析缓冲液以一定比例稀释,每孔加入稀释后的标记抗体150μl。37℃振荡孵育1h,洗涤6次,拍干,加150μl增强液,室温慢速振荡孵育5分钟。然后在PerkinElmer1235分析仪上测定荧光值。
6.最佳的偶联物的确定
分别包被T4-IgG、T4甲酯-IgG和T3-IgG三种偶联物,其包被终浓度均为2μg/ml,进行FT4的TRFIA分析,得到FT4的标准曲线。依据ED50和Bmax来选取最佳偶联物。
7.系统优化：对最佳包被量、标记抗体的最佳稀释度以及最佳反应时间进行确定。
8.自制的TRFIA FT4检测方法的性能评估
8.1标准曲线和分析灵敏度的确定
以X=Ln(浓度),Y=Logit (B/Bmax)=Ln ((B/Bmax)/(1-B/Bmax)),以此绘制标准曲线,计算线性相关性。平行测定十次校准品A的荧光值,并计算其标准差,用A点荧光值的平均值减去2倍标准差得到的荧光值,依据PerkinElmer1235分析仪自带的软件可计算出所对应的浓度,此浓度即为分析灵敏度。
8.2精密度
对低、中、高三个不同浓度的FT4样本进行重复测定10次,计算分析内和分析间变异系数(CV)。
8.3特异性
(一)计算加入的待测交叉反应物质T3,反T3及T2分别能达到50%抑制时的浓度；(二)总T4分析本身达到50%抑制时T4的浓度。
交叉反应定义为：后者与前者的比值×100%。
8.4甲状腺激素相关结合蛋白的抗干扰分析
将一定量的TBG、HSA和Prealbumin加至零点校准品中。最终得到分别含有4个不同终浓度的TBG (20,40,60和100mg/L), HSA (20,40,60和100g/L)and Prealbumin (100,200,300和500mg/L)零点校准品。比较未添加和添加相关蛋白的零点标准品荧光值的变化。
8.5游离脂肪酸的抗干扰分析
将1.1ml不同浓度的水相油酸分别加至PerkinElmer DELFIA FT4试剂盒中的D点冻干校准品中(该试剂盒标示的浓度为15.2pmol/L),充分溶解,最终得到4个含有不同浓度(1,2,3和5mmol/L)的油酸的校准品。以加入1.1ml纯化水溶解的D点冻干校准品的浓度作为对照(当作100%),比较添加油酸后的校准品测定的FT4的浓度值变化。
8.6与进口试剂的相关性比较
用进口PerkinElmer DELFIA FT4试剂盒作为对照方法以评价我们建立的FT4的TRFIA的有效性。110份血清样本同时用两种方法进行检测,计算其相关系数。
(三)血清FT3时间分辨荧光免疫分析法的建立
1.利用活性炭、高岭土制备去激素人血清作为校准品配制用基质。
2.采用T3抗原配制FT3校准品,并用商业化的DELFIA FT3assay进行标定,标定后的校准品浓度依次为：0pmol/L,2.0pmol/L,4.0pmol/L,8.0pmol/L,16pmol/L,32pmol/L。
3.Eu3+标记抗T3多克隆抗体的制备：抗体经预处理后和标记试剂DTTA-Eu3+充分混匀,置于室温过夜,次日上样过分子筛纯化。收集纯化后的目的产物,经0.22μm微孔过滤器过滤,用BCA蛋白定量试剂盒对标记抗体的浓度进行定量,最后加入铕标稳定液至终浓度为0.2%。
4.96孔包被反应板的制备：将已知浓度的偶联物用包被液稀释成某一终浓度,每孔加入150μl,37℃恒温箱中静置20小时。洗板3次后拍干,加入封闭液280μl/孔,4℃静置12h。直接倒去封闭液,拍干。
5. TRFIA标记抗体法检测FT3
每孔加入25μl血清样本或校准品,将标记抗体用分析缓冲液以一定比例稀释,每孔加入稀释后的标记抗体150μl。37℃振荡孵育1h,洗涤6次,拍干,加150μl增强液,室温慢速振荡孵育5分钟。然后在PerkinElmer1235分析仪上测定荧光值。
6.最佳的偶联物的确定
分别包被T2-IgG、T3甲酯-IgG和T3-IgG三种偶联物,其终浓度均为2μg/ml,进行FT3的TRFIA分析,得到FT3标准曲线。依据ED50和Bmax来选取最佳偶联物。
7.系统优化：对最佳包被量、标记抗体的最佳稀释度以及最佳反应时间进行确定。
8.自制的TRFIA FT3检测方法的性能评估
8.1标准曲线和分析灵敏度的确定
以X=Ln(浓度),Y=Logit (B/Bmax)=Ln ((B/Bmax)/(1-B/Bmax),以此绘制标准曲线,计算线性相关性。平行测定十次校准品A的荧光值,并计算其标准差,用A点荧光值的平均值减去2倍标准差得到的荧光值,依据PerkinElmer1235分析仪自带的软件可计算出所对应的浓度,此浓度即为分析灵敏度。
8.2精密度
对低、中、高三个不同浓度的FT3样本进行重复测定10次,计算分析内和分析间变异系数(CV)。
8.3特异性
(一)计算加入的待测交叉反应物质T4,反T3及T2分别能达到50%抑制时的浓度；(二)总T3分析本身达到50%抑制时T3的浓度。
交叉反应定义为：后者与前者的比值×100%。
8.4甲状腺激素相关结合蛋白的抗干扰分析
将一定量的TBG、HSA和Prealbumin加至零点校准品中。最终得到分别含有4个不同终浓度的TBG (20,40,60和100mg/L), HSA (20,40,60和100g/L)and Prealbumin (100,200,300和500mg/L)零点校准品。比较未添加和添加相关蛋白的零点标准品荧光值的变化。
8.5游离脂肪酸的抗干扰分析
将1.1ml不同浓度的水相油酸分别加至PerkinElmer DELFIA FT3试剂盒中的D点冻干校准品中(该试剂盒标示的浓度为9.4pmol/L),充分溶解,最终得到4个含有不同浓度(1,2,3和5mmol/L)的油酸的校准品。以加入1.1ml纯化水溶解的D点冻干校准品的浓度作为对照(当作100%),比较添加油酸后的校准品测定的FT3的浓度值变化。
8.6与进口试剂的相关性比较
用进口PerkinElmer DELFIA FT3试剂盒作为对照方法以评价我们建立的FT3的TRFIA的有效性。110份血清样本同时用两种方法进行检测,计算其相关系数。
结果：
(一)五种甲状腺激素的衍生物的合成和与兔IgG偶联物的制备
成功合成了五种甲状腺激素的衍生物,并分别与兔IgG发生偶联得到五种偶联物T4-IgG、T3-IgG、T4甲酯-IgG、T3甲酯-IgG和T2-IgG。其浓度分别为：1.0mg/ml、1.1mg/ml、0.9mg/ml、1.0mg/ml和1.1mg/ml。
(二)血清FT4时间分辨荧光免疫分析法的建立
1.制备的Eu3+标记抗T4单克隆抗体的浓度为130μg/ml;
2.通过比较T4-IgG、T4甲酯-IgG和T3-IgG三种偶联物产生的灵敏度高低,我们选取了T3-IgG作为最佳的包被偶联物；
3.对包被量、标记抗体的稀释度以及反应时间进行优化分析,得出：最佳的包被量为3μg/ml;标记抗体的最佳稀释度为1：1000；最佳反应时间为1h；
4.FT4的标准曲线方程为：Y=-1.146X+1.324,相关性r=0.9997,计算得到的分析灵敏度为0.2pmol/L;
5.精密度
分析内变异系数为3.5-6.6%,分析间变异系数为和4.4-9.8%,精密度良好。
6.特异性
与T3的交叉反应率为1.5%,与反T3的交叉反应率为1.2%,与T2的交叉反应率小于0.1%。
7.甲状腺激素相关结合蛋白的抗干扰分析
建立的FT4TRFIA对于三种结合蛋白TBG、HSA以及Prealbumin的浓度变化不敏感,基本没有干扰。
8.游离脂肪酸的抗干扰分析
当样本中油酸的浓度为2mmol/L时,基本对本检测方法不会有干扰,但当样本中油酸的浓度升至5mmol/L时,会使FT4的测值偏高15%左右。
9.与PE进口试剂的相关性比较
两种方法均检测110例样本,本研究方法与对照PE试剂盒相关性达到R2=0.984,能够很好的满足临床检测的要求。
(三)血清FT3时间分辨荧光免疫分析法的建立
1.制备的Eu3+标记抗T3单克隆抗体的浓度为150μg/ml；
2.通过比较T2-IgG.T3甲酯-IgG和T3-IgG三种偶联物产生的灵敏度高低,我们选取了T2-IgG作为最佳的包被偶联物；
3.对包被量、标记抗体的稀释度以及反应时间进行优化分析,得出：最佳的包被量为2.5μg/ml;标记抗体的最佳稀释度为1：800；最佳反应时间为1h；
4.FT3的标准曲线方程为：Y=-2.344X+1.980,相关性r=0.9998,计算得到的分析灵敏度为0.3pmol/L；
5.精密度
分析内变异系数为4.4-6.8%,分析间变异系数为4.8-9.6%,精密度良好。
6.特异性
与T4的交叉反应率为0.2%,与反T3的交叉反应率小于0.1%,与T2的交叉反应率为0.4%。
7.甲状腺激素相关结合蛋白的抗干扰分析
建立的FT3TRFIA对于三种结合蛋白TBG.HSA以及Prealbumin的浓度变化不敏感,基本没有干扰。
8.游离脂肪酸的抗干扰分析
当样本中油酸的浓度为2mmol/L时,基本对本检测方法不会有干扰,但当样本中油酸的浓度升至5mmol/L时,会使FT3的测值偏高不到10%。
9.与进口试剂的相关性比较
两种方法均检测110例样本,本研究方法与对照PE试剂盒相关性达到R2=0.966,能够很好的满足临床检测的要求。
结论：
上述结果表明本论文建立的FT4和FT3时间分辨荧光免疫测定方法各项指标(准确性、灵敏度、精密性、特异性、抗干扰性等)均能够满足临床检测试剂要求,可与国外价格高昂的同类试剂相竞争。
Background and objectives
Most 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 fluoroimmunoassay
1. 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 TRFIA
25uL 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 conjugate
T4-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 optimization
The optimal coating concentration, the optimal dilution of labelled antibody and the optimal incubation time were determined.
8. Evaluating the performance of TRFIA for the FT4detection
8.1Calibration 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 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 assay
Precision 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 proteins
Various 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 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 FT4Assay
The 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 fluoroimmunoassay
1. 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 TRFIA
25μL 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 conjugate
T2-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 optimization
The optimal coating concentration, the optimal dilution of labelled antibody and the optimal incubation time were determined.
8. Evaluating the performance of TRFIA for the FT3detection
8.1Calibration 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 assay
Precision 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 proteins
Various 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 FT3Assay
The 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 conjugates
We 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 FT4TRFIA
1. 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. Precision
The intra-assay coefficient of variation was3.5-6.6%and the inter-assay coefficient of variation was4.4-9.8%, bearing good precision.
6. Specificity
Cross-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 proteins
The established FT4TRFIA is not sensitive to the concentration changes of TBG, HSA and Prealbumin, basically reflecting no interference.
8. Interference of free fatty acid
When 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 FT4Assay
Both 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 FT3TRFIA
1. 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. Precision
The intra-assay coefficient of variation was4.4-6.8%and the inter-assay coefficient of variation was4.8-9.6%, bearing good precision.
6. Specificity
Cross-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 proteins
The established FT3TRFIA is not sensitive to the concentration changes of TBG, HSA and Prealbumin, basically reflecting no interference.
8. Interference of free fatty acid
When 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 FT3Assay
Both 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.
Conclusions
These 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.
引文
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[33]Rassaie MJ, Kumari LG, Pandey PK, et al.A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes. Steroids.1992;57(6):288-94
[34]Rao P.N, Taraporewala I.B.A sensitive enzyme-linked immunosorbent assay (ELISA) for testosterone:use of a novel heterologous hapten conjugated to penicillinase.Steroids.1992;57(4):154-61
[35]Mitsuma M, Kambegawa A, Okinaga S, et al. A sensitive bridge heterologous enzyme immunoassay of progesterone using geometrical isomers.J Steroid Biochem.1987;28(1):83-8
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[37]Christofides ND. Free analyte immunoassay.In:Wild D,ed. The Immunoassay Handbook.2nd edn.London:Nature Publishing Group,2001
[38]Stevenson H.P, Archbold G.P.R, Johnston P,et al.Misleading serum free thyroxine results during low molecular weight Heparin treatment. Clin Chem. 1998;44:1002-7
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[41]Mikola H,Miettinen P.Preparation of europium-labeled derivatives of Cortisol for time-resolved fluoroimmunoassays. Steroids.1991;56:17-21
[42]Mikola H,Sundell A.C,Hanninen E.Labeling of estradiol andtestosterone alkyloxime derivatives with a europium chelate for time-resolved fluoroimmunoassays. Steroids.1993;58:330-4
[43]Basu A,Nara S,Chaube S.K,et al.The influence of spacer-containing enzyme conjugateon the sensitivity andspecificity of enzyme immunoassays for hapten. Clin Chim Acta.2006;366:287-92
[44]Snyder S.M,Cavalieri R.R,Goldfine D,et al. Binding of thyroid hormones and their analogues to thyroxine binding globulin. J Biol Chem.1976;251:6489-94
[45]Tsutsumi S,Ishibashi K,Miyai K, etal. A new radioimmunoassay of free thyroxin using 125I-labeled thyroxine-protein complex uninfluenced by albumin and thyroxin-binding globulin. Clin Chim Acta 1987; 170:315-22
[46]Nicolson R.E,Reilly C.P,Pannall P.R,et al. Do nonesterified fatty acids displace thyroxin from its plasma binding sites in severe nonthyroidal illnesses? Clin Chem.1989;35:931-4
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