睾酮时间分辨荧光免疫检测试剂及降钙素原胶体金半定量免疫层析检测试剂的研究
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摘要
研究背景及目的
免疫标记技术指用荧光素、放射性同位素、酶、铁蛋白、胶体金及化学(或生物)发光剂等作为示踪物,标记抗体或抗原进行的抗原抗体反应,并借助于荧光显微镜、射线测量仪、酶标检测仪、电子显微镜和发光免疫测定仪等精密仪器,应用各种液相和固相免疫分析方法,对体液中的半抗原、抗原或抗体进行定性和定量测定的方法。
时间分辨荧光免疫分析(Timed-resolved fluoroimmunoassay, TRFIA)是一种灵敏的高端定量免疫检测技术,是以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十分广泛等优点。时间分辨荧光免疫分析相对于酶免分析、放射免疫分析有更高的临床应用价值。该技术适用于各种抗原抗体的临床检测,在市场上已得到广泛应用。
睾酮作为性激素的一种,是临床激素六项检测中的重要组成部分,目前临床上常用的睾酮检测试剂主要是PE公司生产的时间分辨睾酮检测试剂及进口的化学发光法睾酮检测试剂,于是开发国产的睾酮检测试剂成为我们关注的焦点。睾酮由于是甾类激素,属小分子半抗原,98%的睾酮分子在血清中均是以与激素结合蛋白结合的状态存在,这些特殊性均为睾酮定量检测试剂的研发工作带来了困难与挑战。因此,在睾酮试剂的研制过程中,我们将异源桥式搭配理念引入研制过程中,以睾酮的结构类似物雄烯二酮为载体,在其C3位引入含有五个亚甲基的臂,在此结构基础上合成NHS活泼酯,其与BSA偶联后进行Eu标记从而作为反应体系中的示踪物,在以一步间接竞争法的模式基础上研制了此试剂用于血清、血浆中睾酮含量的测定。
胶体金免疫层析是建立在酶联免疫吸附试验、乳胶凝集试验、单克隆抗体技术和免疫胶体金标记技术基础上的以胶体金为标记物,利用特异的抗原抗体反应来放大反应,通过直接观察就可以判定结果的新技术。该技术具有简单、快速、准确和无污染等优点,在临床医学检测、激素检测、食品安全检测、药物残留和毒品快速检测诸多诊断领域迅速发展[1]。随着即时检验(POCT)产业的深入发展,胶体金试剂由于其本身所带有的快速、简便、易于携带、便于深入社区及边远山区等特点,成为目前POCT发展的主要领域,从而那些与发病急、危害大的疾病相关的检测指标的胶体金试剂的需求越来越大。
降钙素原(PCT)作为一种与脓毒血症相关的特异性检测指标,只有当细菌感染时降钙素原才会升高,在其他的炎性反应中如病毒感染、自身免疫性疾病和创伤等均不会升高,所以其具有良好的特异性及灵敏度,能用于区分感染或非感染引起的炎症、器官功能障碍以及休克。从产生和消除的动力学过程来看,PCT也是最适合用作临床诊断指标的。加之脓毒血症能够在短时间内对病人的生命造成严重威胁,故其检测试剂的胶体金化也成为临床医生及诊断试剂厂家的关注的重点。但是目前检验领域仅有几家公司有相关试剂的生产能力,国内厂家几乎属于空白状态,大部分医院所使用的相关检测试剂全部依赖进口,如Braham公司生产的半定量PCT试剂,但其所具有的一个重要的缺陷是价格昂贵,无法满足社区与边远山区医疗需求,故研制国产的PCT胶体金半定量试剂成为我们的主要目标。国产PCT试剂一直寥寥无几的主要原因是没有性能优越的原料,大部分存在灵敏地低或特异性差的问题。为了更好的解决原料问题,我们对市售的PCT抗体进行了大规模的搜索工作,利用时间分辨免疫荧光检测进行原料配对等初筛工作,并且发现了满足半定量胶体金试剂研发要求的原料在TRFIA荧光值方面的特殊性,最终通过双抗体夹心法研制了能够进行PCT半定量检测的胶体金试剂。
本文主要从制备睾酮及睾酮类似物的化学衍生物及其偶联物、睾酮时间分辨免疫荧光检测试剂的研究及降钙素原半定量检测试剂的研究共计三个方面对所进行的研究做一总结。
第一部分内容中,着重介绍了睾酮及睾酮类似物的化学衍生物及其偶联物的制备,制备过程以睾酮或睾酮类似物-雄烯二酮作为合成起始原料,向上述结构中引入O-(羧甲基)羟胺半盐酸盐(O-(carboxymethyl)hydroxylamine hemi-hydrochloride)或氨氧基已酸氢溴酸盐(6-Aminooxy-hexanoic acid,hydrobromide)作为臂结构。随后在NHS及DCC的条件下,搅拌过夜,将相应的肟合成NHS活泼酯结构,利用闪氏柱层析对其进行纯化,除去未反应的成分。最后将将活泼酯与BSA以摩尔比15：1的比例进行偶联,反应于室温下搅拌过夜,次日以分子筛进行纯化,并用BCA法分别检测四种睾酮偶联物Ⅰ、Ⅱ、Ⅲ及Ⅳ的浓度分别为0.8mg/ml、0.9mg/ml、0.9mg/ml及0.8mg/ml。
第二部分着重介绍了人睾酮时间分辨荧光免疫分析检测试剂的研究过程。首先利用高岭土、活性炭制备去激素血清作为校准品配制用基质,并配制睾酮校准品如下：A(0nmol/L)、B(1.0nmol/L)、C(3.2nmol/L)、D(6.8nmol/L)、E(14nmol/L)及F(51.2nmol/L)。第二步进行睾酮偶联物的标记,每种偶联物都取1mg进行标记,按照偶联物与Eu3+质量比2：1的比例加入铕标记配体,标记体系均为200μl,将待标记偶联物和铕标记试剂充分混匀后置于摇床上室温孵育18h。次日过分子筛层析纯化。根据280nm蛋白的吸收峰收集洗脱液,即为睾酮检测原。利用BCA法检测睾酮检测原的浓度,然后向其中加入铕标保护液,置—20℃保存。最终检测到睾酮检测原Ⅰ、Ⅱ、Ⅲ及Ⅳ的浓度分别为132μg/ml、135μg/ml、138μg/ml和140μg/ml。第三步是摸索羊抗鼠IgG的最适包被浓度,设置包被梯度,用包被液将羊抗鼠IgG多抗稀释至不同浓度后进行,37℃过夜,封闭,真空抽干,—20℃冷冻保存,检测在不同的包被浓度下,睾酮校准品A的荧光值,通过荧光值的比较选择最适的羊抗鼠IgG包被浓度,最终确定羊抗鼠IgG的最适包被浓度为6μg/ml。第四步是进行四种睾酮检测原的性能比较,根据各个睾酮检测原的标准曲线分别确定其ED50,选择ED50最低的检测原作为体系中所使用的示踪物,最终确定睾酮检测原Ⅳ为最佳检测原。第五步确定最适的样本加入量,将样本加入量做梯度,检测在不同加入量条件下的校准品A的荧光值(Bmax)及校准品B的荧光值,并计算B/Bmax的比值,选择比值相对较小并且不易对操作引入误差的样本量,最终确定最适样本加入量为25μl。第六步确定抗睾酮单克隆抗体及检测原的最佳使用浓度,将二者的浓度进行梯度稀释,利用棋盘滴定法进行实验,根据灵敏度及A点荧光值来选择最适二者最适的稀释度,最终确定抗睾酮单克隆抗体的最佳使用浓度为6ng/ml,检测原的最佳使用浓度为1μg/ml。由于血清中99%的睾酮是以与SHBG(性激素结合蛋白)相结合的形式存在,故反应缓冲液中阻断剂的存在对于血清睾酮含量的准确测定具有重要的意义,于是第七步我们进行阻断剂最佳浓度的测定,将二甲氧雌二醇的浓度做梯度稀释,根据不同阻断剂浓度下A点的荧光值及各个点的B/Bmax进行比较,选择灵敏度比较高且阻断剂的加入对于A点荧光值的影响程度在5%以内的浓度为阻断剂最适使用浓度,最终确定阻断剂的最适使用浓度为30μg/ml。第八步进行最佳反应时间的确定,将反应时间做梯度,检测不同反应时间下校准品A、B、C、D、E和F的荧光值与Btotal的比值,以各个校准品的反应都趋于平衡的时间作为体系的最佳反应时间,最终将最适反应时间定为90分钟。待试剂的各个参数都确定完毕后,对试剂的整体性能进行评价,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,将得到的荧光值带入标准曲线,所得的浓度即为分析灵敏度。经计算,分析灵敏度为0.15nmol/L;取不同浓度三个样本分别进行梯度稀释,不同稀释度条件下的测量浓度与理论浓度的比值即为稀释回收率,此参数反映体系的健全性,本试剂的稀释回收率在98.8%-101.4%之间,稀释倍数与含量呈线性关系,说明本分析具有良好的健全性；研究此反应的特异性,将临床上可能与睾酮检测有交叉反应的各类激素样本在睾酮检测体系中进行检测,取睾酮体系ED50所对应的睾酮浓度与交叉物质在睾酮体系中达到A点荧光值一半时的浓度的比值,即为二者之间的交叉反应率,经实验证实,本试剂与孕酮的交叉反应率为0.14%,与双氢睾酮的交叉反应率为0.38%,与雌二醇的交叉反应率为0.07%,与17-羟基孕酮的交叉反应率为0.02%,与雌三醇的交叉反应率为0.09%,与Danazol的交叉反应率为0.12%；在精密度分析方面,采用三个伯乐质控品,各设10个复孔,同一次实验内和不同实验中重复多次测定,分析内变异系数为2.5-6.6%,分析间变异系数为3.6-7.8%,精密度良好。以SPSS13.0统计软件对数据进行分析,在线性评估方面应用直线回归分析方法,以P＜0.05为差异有统计学意义。利用Logit函数将睾酮的S形竞争曲线转换为直线,以此直线的相关系数评价体系的性能,转换为Logit函数后,线性回归方程为Y=1.453-2.193X,相关系数为r=0.99,R2=1.000,F=7303.334,P＜0.001,说明此竞争法体系的线性关系非常好；最后将本试剂与PE试剂做相关分析,分别对84例样本进行检测,评价两种试剂之间的检测相关性,相关性方程为Y=0.991X-0.331,相关系数为r=0.988,R2为0.977,F=3407.808,P＜0.001,说明本试剂能够很好的满足临床检测的要求。
本论文的第三个部分主要阐述了降钙素原胶体金半定量免疫层析检测试剂的研制过程。研制过程中,首先进行NC膜的筛选,对几种候选NC膜通过毛细管流时、灵敏度、反应结果出现时间及试剂条背景等指标的比较确定本试剂最终用膜,最终选取Millipore公司生产的HF135作为本试剂用膜；第二步进行的是胶体金结合垫材质的筛选,对候选的胶体金用玻璃纤维通过材质、非特异性吸附、胶体金能否恒定释放、金标垫上胶体金溶解与释放速度及能否预防溶血等方面进行比较后确定所选取的材质,经过一系列实验,最终确定选择Millipore的玻璃纤维GFCP103000作为本试剂用的胶体金结合垫材质；研制过程的第三步是原料的筛选过程,主要是通过软件SPSS13.0对TRFIA数据进行线性回归分析,以P<0.05为差异有统计学意义,计算r值及P值,经分析,组合1至组合9均表现为线性性差,r值分别为0.982、0.958、0.973、0.966、0.786、0.859、0.884、0.977及0.458,P值分别为0.003、0.01、0.005、0.007、0.214、0.062、0.047、0.004及0.438,组合10及组合11经分析线性性好,r值分别为0.991及0.993,P值均为0.001。其后再利用胶体金方法对2对初筛得到的抗体组合进行进一步评估,评价配对后的特异性、灵敏度、比色区分度等指标,确定最佳配对,经过严密的实验设计及严谨的实验过程,最终确定使用Hytest的16B5作为包被原料,杭州启泰的MJG03作为标记原料。第四步是对所确定的标记原料进行最适标记条件的摸索,利用氯化钠法确定原料的最适标记pH值和最适标记量,经实验可知,MJGO3的最适标记pH条件为1ml金中加入0.1M的K2CO315μl,标记量为每100ml胶体金溶液标记1.5mg。第五步是确定包被用原料的包被条件,通过设置不同的包被条件,检测在不同包被条件下试剂的灵敏度、特异性、T线显色程度等指标,选择T线性能最好的条件,最终选择10mM PBS(pH7.2)作为包被液。第六步进行金标抗体使用浓度及包被浓度的摸索,通过将喷金量及包被浓度做梯度,选择能够同时满足灵敏度高、特异性好且0.5ng/ml、2ng/ml及10ng/m1三个浓度显色差异大的组合,经过棋盘滴定法实验,最终确定金标抗体的使用浓度为OD535=40,喷金量为0.5μl/mm,检测线抗体16B5的工作浓度为2.5mg/ml,划线量为0.12μl/mm;质控线抗体的最佳工作浓度为1mg/ml,划线量为0.12μl/mm。在确定最佳喷金量及包被浓度的前提下,第七步进行比色卡标准的建立,将0.5ng/ml、2ng/ml及10ng/ml三个浓度的阳性样本各测定20次,分别选择20次结果的平均显色度作为比色卡的标准。所有试剂相关的参数都确定后,对试剂性能进行进一步评估,通过测定检测不同降钙素原浓度的样本,最终确定试剂的最低检出量为0.2ng/ml;利用C-反应蛋白、降钙素、类风湿因子阳性样本评价试剂的特异性,发现本试剂与10mg/m1的C-反应蛋白、6.4mg/m1的降钙素及500IU/m1的类风湿因子均无交叉反应,特异性良好；血清血浆样本检测结果一致性考核方面,将PCT标本分别处理成血清、肝素抗凝血浆、EDTA抗凝血浆样本,确定血清、血浆检测结果是否一致,最终通过实验发现试剂在这两种样本的检测性能上具有一致性；本试剂经稳定性评价发现其在37℃条件下放置21天不会对试剂性能造成影响：精密性评价是平行测定0.5ng/ml、2ng/ml及10ng/ml参考品各十次,观察其显色程度,通过平行实验发现,本试剂精密性良好；最后将本试剂与定量试剂盒进行比较,取临床定量检测样本45例,用本试剂进行半定量检测,将二者的结果进行对比,以评价试剂半定量的准确性,通过实验发现,二者的符合率为88.9%。
Immunolabelling technologies are method for qualitative and quantitative determ ination of the body fluid of the hapten, antigen or antibody. The antigen antibody reac tion was labeled antibody or antigen by using of fluorescein, radioactive isotope, enzy me, ferritin, colloidal gold and chemical (or biological) luminescence agent as tracer. These methods also with the help of fluorescence microscopy, X-ray measuring instru ment, ELISA test, electron microscopy and luminescence immunoassay instrument pr ecision application of various instruments, liquid phase and solid phase immunoassay m-ethod.
Time-resolved fluorescence immunoassay (Timed-resolved fluoroimmunoassay, TRFIA) is an advanced sensitive quantitative immunoassay technology. The technolo gy is with lanthanide ions as a marker to mark the antigen or antibody. Because of Ian thanide ions have unique fluorescence properties, the technology can achieve high sig nal-to-noise ratio, thus has high sensitivity, and has the advantages of simple preparati on, markers of storage time, free of radioactive pollution, detection reproducibility, sh ort operating process, the standard curve range is wide, not affected by sample natural fluorescence interference and the application range is very extensive. Time-resolved fluorescence immunoassay compared with enzyme immunoassay, radioimmunoassay has higher clinical application value. Clinical detection of the technology is suitable f or all kinds of antigen and antibody, has been widely used in the market.
Testosterone is a hormone, which detection is one important part of clinically six h-ormone detection. Clinically used in testosterone detection reagent mainly are the c he-miluminescence detection reagent which imported and time-resolved testosterone assay kit from testosterone production company PE currently. Testosterone detection reagent and the development of domestic become the focus of our attention. Testoster one as the steroid hormone, is a small molecular hapten molecules in serum testostero ne,98%are present in combination with hormone binding protein, which has brought the difficulty and challenge these particularities to testosterone ELISA R&D work. Therefore, in the development process of testosterone reagent, we will introduce the i dea of heterologous bridge with the development process, structural analogues of test osterone androstene two ketone as the carrier, in its C3introduction containing five m ethylene arm, on the basis of the structure of synthetic NHS active ester, and BSA co-upling by Eu markers thus as a tracer in the reaction system. The indirect competition method was developed for determination of the reagent, serum testosterone levels in plasma.
Colloidal gold immune chromatography is a new technology based on ELISA, la-tex agglutination test, monoclonal antibodies and immune colloidal gold labeling tec-hnique, which is marked by colloidal gold, to amplify the response using the antigen antibody reaction specificity. It can be judged by direct observation. This technology has the advantages of simple, rapid, accurate and no pollution. Development of variou-s diagnosis field in clinical medicine detection, hormone detection, detection of food safety, drug residue rapid detection and drug fast [1]. With the development of instant test (POCT) in-depth, colloidal gold reagent has become the main field for the develo pment of POCT at present because of its fast, convenient, with easy to carry, easy to i-ndepth community and remote and a mountainous area, so the requirement of colloid-al gold reagent for detecting acute onset, the harm of diseases related became urgenc-y.
This article is composed by three parts.
In the first part, four kinds of testosterone and testosterone analogues chemical derivatives and conjugates prepared. In the produce, with testosterone or testosterone analogues-androstene as raw material, then introduced O-(carboxymethyl) hydroxylamine hydrochloride (O-(carboxymethyl) hydroxylamine hemi-hydrochloride) or aminooxy acetic acid hydrobromide (6-Aminooxy-hexanoic acid, hydrobromide) as the arm structure. In the next step, the active ester was synthesized as following:in the NHS and DCC conditions, stirring overnight, the oxime synthesis of NHS corresponding to the active ester structure, use flash's chromatography to remove unreacted components. Then took active ester and BSA scale with the molar ratio of15:1coupling reaction at room temperature, stirring overnight, the next day to molecular sieve purification, and four types of conjugates concentrations were detected by BCA method. At last, concentrations of testosterone conjugates I, II, III and IV were0.8mg/ml、0.9mg/ml、0.9mg/ml and0.8mg/ml.
In the second part, we described the development of testosterone time-resolved fluorescence immunoassay reagent. First, steroid-free serum was prepared with kaolin and activated carbon, and calibrator was prepared with the steroid-free serum just as following:A(0nmol/L)、B (1.0nmol/L)、C(3.2nmol/L)、D(6.8nmol/L)、 E(14nmol/L)and F(51.2nmol/L). The next step is the labelling of testosterone conjugates. Mixed each conjugate1mg with0.5mg Eu3+and incubated for18h at room temperature. The product was purified and detect the testosterone concentration by BCA assay, then the Eu standard solution was added to the purified product and preserved under-20℃.The concentration of testosterone tracers were132μg/ml、135μg/ml、138μg/ml和140μg/ml separately. Step three is the definition of the optimum coating concentration of goat anti-mouse IgG. Detected and compared the fluorescence value of testosterone calibrator A in different coating concentration separately, the optimal goat anti-mouse IgG coating concentration is6μg/ml. Step four is the comparison of four testosterone tracers. Detected the ED50of each testosterone tracer, and chose tracer IV with the lowest ED50as the optimal testosterone tracer. Step five is the definition of sample quantity. Detected the fluorescence value of testosterone calibrator A (Bmax) and the fluorescence value of testosterone calibrator B under different sample quantity separately, calculated the B/Bmax ratio, chose25μl with the lower B/Bmax ratio and the easier operation procedure as the optimal sample amount. Step six is the definition of optimal concentration of testosterone monoclonal antibody and testosterone tracer. The srandard of determination is sensitivity and fluorescence value of testosterone calibrator A, finally the optimal concentration of testosterone monoclonal antibody is6ng/ml, the optimal concentration of testosterone tracer is1μg/ml. In human serum,99%of testosterone is bind with SHBG, so the blocking agent plays important role in the exact detection of testosterone. Step seven is the determination of optimum concentration of blocking agent. Detected the fluorescence level of testosterone calibrator, calculated and compared the B/Bmax of each calibrator in different concentration of blocking agent, evaluated the sensitivity and influence of blocking agent concentration on the reaction system, determined the optimal concentration of blocking agent was30μg/ml. Clarifyed the optimum reaction time in step eight, detected the fluorescence ratio with calibrator A, B, C, D, E,F and Btotal in different reaction time, eventually optimal reaction time is90minutes.Overall evaluation of the reagent was carried out after all the parameters were determined. Analytical sensitivity is0.15nmol/L; The recovery rate of dilution is between98.8%and101.4%, there is a linear relationship between the dilution and concentration. Then analysis the specificity of the reagent, the cross-reactivity of progesterone was0.14%, the cross-reactivity of DHT was0.38%, the cross-reactivity of estradiol was0.07%, the cross-reactivity of17-hydroxyl progesterone was0.02%, and the cross-reactivity of estriol was0.09%, and the cross-reactivity of Danazol was0.12%. In the aspect of precision analysis, the coefficient of variation (CVs) was2.5-6.6%(intraassay)and3.6%～7.8%(interassay). In terms of the evaluation of linear, linear formula is Y=1.453-2.193X, the correlation coefficient r=0.99, R2=1.000,F=7303.334,P<0.001, shows that the linearity of this competition system is perfect; Compared with PE reagent kit,84samples were detected, correlation formula was Y=0.991X-0.331, the correlation coefficient was0.988, R2=0.977, F=3407.808, P<0.001, the result reflected that this reagent can meet the requirements of clinical detection very well.
The third part of the thesis mainly introduces the development process of procalcitonin colloidal gold immunochromatography semi-quantitative detection.In the reagent development process, the NC membrane was screened at first, HF135was determined by the comparison of capillary flow, sensitivity, response time and the background; the second step is the definition of the colloidal gold combined pad material, The selection of colloidal gold candidate materials were compared from glass fiber materials, the nonspecific adsorption, colloidal gold constant release, colloidal gold dissolution and release rate, and hemolysis prevention. The final determination is GFCP103000produced by Millipore after a series of experiments; the third step is the screening of raw materials by TRFIA and colloidal gold method. Evaluated29combinations of antibodies successively, contain pairing specificity, sensitivity, color discrimination, and so on. Ultimately,16B5of Hytest was determined as coating material, MJG03of Hangzhou Qi Tai was determined as labeling material. The fourth step is definition of the optimal labeling condition of MJG03, the optimum labeling conditions of MJG03is15μl0.1M K2CO3per milliliter of colloidal gold, and1.5mg MJG03every100ml colloidal gold. The fifth step is to determine the best coating condition of16B5, lOmM PBS (pH7.2) was chosen as the coating buffer by setting different packages. Detection in different coating conditions reagent sensitivity, specificity, T line color degree conditions, choose the best performance of T line. The sixth step is the determination of the concentration of16B5and gold-labeled MJG03, ultimately the concentration of gold-labeled MJG03is OD535=40, the spraying quantity is0.5μl/mm, the concentration of16B5is2.5mg/ml, the spraying quantity is0.12μl/mm, the optimal concentration of goat anti mouse IgG is lmg/ml, the spraying quantity is0.12μl/mm. The seventh step is the establishment of standard, the positive samples of0.5ng/ml,2ng/ml and lOng/ml were measured for20times separately, and then took the average chromaticity as color standard. The evaluation of reagent carried out after related parameters were determined, the sensitivity of reagent is0.2ng/ml, and the reagent did not react with C-reactive protein(10mg/ml), calcitonin(6.4mg/ml), and rheumatoid factor(500IU/ml). The consistency of plasma and serum is perfect. The performance of reagent did not changed under the condition of37℃for21days. The precision of reagent was evaluated by samples with the concentration of0.5ng/ml,2ng/ml and10ng/ml for ten times, and this experiment showed that precision of reagent was perfect. Finally, the reagent was compared with other quantitative reagent, the correlation coefficient is88.9%.
免疫标记技术指用荧光素、放射性同位素、酶、铁蛋白、胶体金及化学(或生物)发光剂等作为示踪物,标记抗体或抗原进行的抗原抗体反应,并借助于荧光显微镜、射线测量仪、酶标检测仪、电子显微镜和发光免疫测定仪等精密仪器,应用各种液相和固相免疫分析方法,对体液中的半抗原、抗原或抗体进行定性和定量测定的方法。
时间分辨荧光免疫分析(Timed-resolved fluoroimmunoassay, TRFIA)是一种灵敏的高端定量免疫检测技术,是以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十分广泛等优点。时间分辨荧光免疫分析相对于酶免分析、放射免疫分析有更高的临床应用价值。该技术适用于各种抗原抗体的临床检测,在市场上已得到广泛应用。
睾酮作为性激素的一种,是临床激素六项检测中的重要组成部分,目前临床上常用的睾酮检测试剂主要是PE公司生产的时间分辨睾酮检测试剂及进口的化学发光法睾酮检测试剂,于是开发国产的睾酮检测试剂成为我们关注的焦点。睾酮由于是甾类激素,属小分子半抗原,98%的睾酮分子在血清中均是以与激素结合蛋白结合的状态存在,这些特殊性均为睾酮定量检测试剂的研发工作带来了困难与挑战。因此,在睾酮试剂的研制过程中,我们将异源桥式搭配理念引入研制过程中,以睾酮的结构类似物雄烯二酮为载体,在其C3位引入含有五个亚甲基的臂,在此结构基础上合成NHS活泼酯,其与BSA偶联后进行Eu标记从而作为反应体系中的示踪物,在以一步间接竞争法的模式基础上研制了此试剂用于血清、血浆中睾酮含量的测定。
胶体金免疫层析是建立在酶联免疫吸附试验、乳胶凝集试验、单克隆抗体技术和免疫胶体金标记技术基础上的以胶体金为标记物,利用特异的抗原抗体反应来放大反应,通过直接观察就可以判定结果的新技术。该技术具有简单、快速、准确和无污染等优点,在临床医学检测、激素检测、食品安全检测、药物残留和毒品快速检测诸多诊断领域迅速发展[1]。随着即时检验(POCT)产业的深入发展,胶体金试剂由于其本身所带有的快速、简便、易于携带、便于深入社区及边远山区等特点,成为目前POCT发展的主要领域,从而那些与发病急、危害大的疾病相关的检测指标的胶体金试剂的需求越来越大。
降钙素原(PCT)作为一种与脓毒血症相关的特异性检测指标,只有当细菌感染时降钙素原才会升高,在其他的炎性反应中如病毒感染、自身免疫性疾病和创伤等均不会升高,所以其具有良好的特异性及灵敏度,能用于区分感染或非感染引起的炎症、器官功能障碍以及休克。从产生和消除的动力学过程来看,PCT也是最适合用作临床诊断指标的。加之脓毒血症能够在短时间内对病人的生命造成严重威胁,故其检测试剂的胶体金化也成为临床医生及诊断试剂厂家的关注的重点。但是目前检验领域仅有几家公司有相关试剂的生产能力,国内厂家几乎属于空白状态,大部分医院所使用的相关检测试剂全部依赖进口,如Braham公司生产的半定量PCT试剂,但其所具有的一个重要的缺陷是价格昂贵,无法满足社区与边远山区医疗需求,故研制国产的PCT胶体金半定量试剂成为我们的主要目标。国产PCT试剂一直寥寥无几的主要原因是没有性能优越的原料,大部分存在灵敏地低或特异性差的问题。为了更好的解决原料问题,我们对市售的PCT抗体进行了大规模的搜索工作,利用时间分辨免疫荧光检测进行原料配对等初筛工作,并且发现了满足半定量胶体金试剂研发要求的原料在TRFIA荧光值方面的特殊性,最终通过双抗体夹心法研制了能够进行PCT半定量检测的胶体金试剂。
本文主要从制备睾酮及睾酮类似物的化学衍生物及其偶联物、睾酮时间分辨免疫荧光检测试剂的研究及降钙素原半定量检测试剂的研究共计三个方面对所进行的研究做一总结。
第一部分内容中,着重介绍了睾酮及睾酮类似物的化学衍生物及其偶联物的制备,制备过程以睾酮或睾酮类似物-雄烯二酮作为合成起始原料,向上述结构中引入O-(羧甲基)羟胺半盐酸盐(O-(carboxymethyl)hydroxylamine hemi-hydrochloride)或氨氧基已酸氢溴酸盐(6-Aminooxy-hexanoic acid,hydrobromide)作为臂结构。随后在NHS及DCC的条件下,搅拌过夜,将相应的肟合成NHS活泼酯结构,利用闪氏柱层析对其进行纯化,除去未反应的成分。最后将将活泼酯与BSA以摩尔比15：1的比例进行偶联,反应于室温下搅拌过夜,次日以分子筛进行纯化,并用BCA法分别检测四种睾酮偶联物Ⅰ、Ⅱ、Ⅲ及Ⅳ的浓度分别为0.8mg/ml、0.9mg/ml、0.9mg/ml及0.8mg/ml。
第二部分着重介绍了人睾酮时间分辨荧光免疫分析检测试剂的研究过程。首先利用高岭土、活性炭制备去激素血清作为校准品配制用基质,并配制睾酮校准品如下：A(0nmol/L)、B(1.0nmol/L)、C(3.2nmol/L)、D(6.8nmol/L)、E(14nmol/L)及F(51.2nmol/L)。第二步进行睾酮偶联物的标记,每种偶联物都取1mg进行标记,按照偶联物与Eu3+质量比2：1的比例加入铕标记配体,标记体系均为200μl,将待标记偶联物和铕标记试剂充分混匀后置于摇床上室温孵育18h。次日过分子筛层析纯化。根据280nm蛋白的吸收峰收集洗脱液,即为睾酮检测原。利用BCA法检测睾酮检测原的浓度,然后向其中加入铕标保护液,置—20℃保存。最终检测到睾酮检测原Ⅰ、Ⅱ、Ⅲ及Ⅳ的浓度分别为132μg/ml、135μg/ml、138μg/ml和140μg/ml。第三步是摸索羊抗鼠IgG的最适包被浓度,设置包被梯度,用包被液将羊抗鼠IgG多抗稀释至不同浓度后进行,37℃过夜,封闭,真空抽干,—20℃冷冻保存,检测在不同的包被浓度下,睾酮校准品A的荧光值,通过荧光值的比较选择最适的羊抗鼠IgG包被浓度,最终确定羊抗鼠IgG的最适包被浓度为6μg/ml。第四步是进行四种睾酮检测原的性能比较,根据各个睾酮检测原的标准曲线分别确定其ED50,选择ED50最低的检测原作为体系中所使用的示踪物,最终确定睾酮检测原Ⅳ为最佳检测原。第五步确定最适的样本加入量,将样本加入量做梯度,检测在不同加入量条件下的校准品A的荧光值(Bmax)及校准品B的荧光值,并计算B/Bmax的比值,选择比值相对较小并且不易对操作引入误差的样本量,最终确定最适样本加入量为25μl。第六步确定抗睾酮单克隆抗体及检测原的最佳使用浓度,将二者的浓度进行梯度稀释,利用棋盘滴定法进行实验,根据灵敏度及A点荧光值来选择最适二者最适的稀释度,最终确定抗睾酮单克隆抗体的最佳使用浓度为6ng/ml,检测原的最佳使用浓度为1μg/ml。由于血清中99%的睾酮是以与SHBG(性激素结合蛋白)相结合的形式存在,故反应缓冲液中阻断剂的存在对于血清睾酮含量的准确测定具有重要的意义,于是第七步我们进行阻断剂最佳浓度的测定,将二甲氧雌二醇的浓度做梯度稀释,根据不同阻断剂浓度下A点的荧光值及各个点的B/Bmax进行比较,选择灵敏度比较高且阻断剂的加入对于A点荧光值的影响程度在5%以内的浓度为阻断剂最适使用浓度,最终确定阻断剂的最适使用浓度为30μg/ml。第八步进行最佳反应时间的确定,将反应时间做梯度,检测不同反应时间下校准品A、B、C、D、E和F的荧光值与Btotal的比值,以各个校准品的反应都趋于平衡的时间作为体系的最佳反应时间,最终将最适反应时间定为90分钟。待试剂的各个参数都确定完毕后,对试剂的整体性能进行评价,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,将得到的荧光值带入标准曲线,所得的浓度即为分析灵敏度。经计算,分析灵敏度为0.15nmol/L;取不同浓度三个样本分别进行梯度稀释,不同稀释度条件下的测量浓度与理论浓度的比值即为稀释回收率,此参数反映体系的健全性,本试剂的稀释回收率在98.8%-101.4%之间,稀释倍数与含量呈线性关系,说明本分析具有良好的健全性；研究此反应的特异性,将临床上可能与睾酮检测有交叉反应的各类激素样本在睾酮检测体系中进行检测,取睾酮体系ED50所对应的睾酮浓度与交叉物质在睾酮体系中达到A点荧光值一半时的浓度的比值,即为二者之间的交叉反应率,经实验证实,本试剂与孕酮的交叉反应率为0.14%,与双氢睾酮的交叉反应率为0.38%,与雌二醇的交叉反应率为0.07%,与17-羟基孕酮的交叉反应率为0.02%,与雌三醇的交叉反应率为0.09%,与Danazol的交叉反应率为0.12%；在精密度分析方面,采用三个伯乐质控品,各设10个复孔,同一次实验内和不同实验中重复多次测定,分析内变异系数为2.5-6.6%,分析间变异系数为3.6-7.8%,精密度良好。以SPSS13.0统计软件对数据进行分析,在线性评估方面应用直线回归分析方法,以P＜0.05为差异有统计学意义。利用Logit函数将睾酮的S形竞争曲线转换为直线,以此直线的相关系数评价体系的性能,转换为Logit函数后,线性回归方程为Y=1.453-2.193X,相关系数为r=0.99,R2=1.000,F=7303.334,P＜0.001,说明此竞争法体系的线性关系非常好；最后将本试剂与PE试剂做相关分析,分别对84例样本进行检测,评价两种试剂之间的检测相关性,相关性方程为Y=0.991X-0.331,相关系数为r=0.988,R2为0.977,F=3407.808,P＜0.001,说明本试剂能够很好的满足临床检测的要求。
本论文的第三个部分主要阐述了降钙素原胶体金半定量免疫层析检测试剂的研制过程。研制过程中,首先进行NC膜的筛选,对几种候选NC膜通过毛细管流时、灵敏度、反应结果出现时间及试剂条背景等指标的比较确定本试剂最终用膜,最终选取Millipore公司生产的HF135作为本试剂用膜；第二步进行的是胶体金结合垫材质的筛选,对候选的胶体金用玻璃纤维通过材质、非特异性吸附、胶体金能否恒定释放、金标垫上胶体金溶解与释放速度及能否预防溶血等方面进行比较后确定所选取的材质,经过一系列实验,最终确定选择Millipore的玻璃纤维GFCP103000作为本试剂用的胶体金结合垫材质；研制过程的第三步是原料的筛选过程,主要是通过软件SPSS13.0对TRFIA数据进行线性回归分析,以P<0.05为差异有统计学意义,计算r值及P值,经分析,组合1至组合9均表现为线性性差,r值分别为0.982、0.958、0.973、0.966、0.786、0.859、0.884、0.977及0.458,P值分别为0.003、0.01、0.005、0.007、0.214、0.062、0.047、0.004及0.438,组合10及组合11经分析线性性好,r值分别为0.991及0.993,P值均为0.001。其后再利用胶体金方法对2对初筛得到的抗体组合进行进一步评估,评价配对后的特异性、灵敏度、比色区分度等指标,确定最佳配对,经过严密的实验设计及严谨的实验过程,最终确定使用Hytest的16B5作为包被原料,杭州启泰的MJG03作为标记原料。第四步是对所确定的标记原料进行最适标记条件的摸索,利用氯化钠法确定原料的最适标记pH值和最适标记量,经实验可知,MJGO3的最适标记pH条件为1ml金中加入0.1M的K2CO315μl,标记量为每100ml胶体金溶液标记1.5mg。第五步是确定包被用原料的包被条件,通过设置不同的包被条件,检测在不同包被条件下试剂的灵敏度、特异性、T线显色程度等指标,选择T线性能最好的条件,最终选择10mM PBS(pH7.2)作为包被液。第六步进行金标抗体使用浓度及包被浓度的摸索,通过将喷金量及包被浓度做梯度,选择能够同时满足灵敏度高、特异性好且0.5ng/ml、2ng/ml及10ng/m1三个浓度显色差异大的组合,经过棋盘滴定法实验,最终确定金标抗体的使用浓度为OD535=40,喷金量为0.5μl/mm,检测线抗体16B5的工作浓度为2.5mg/ml,划线量为0.12μl/mm;质控线抗体的最佳工作浓度为1mg/ml,划线量为0.12μl/mm。在确定最佳喷金量及包被浓度的前提下,第七步进行比色卡标准的建立,将0.5ng/ml、2ng/ml及10ng/ml三个浓度的阳性样本各测定20次,分别选择20次结果的平均显色度作为比色卡的标准。所有试剂相关的参数都确定后,对试剂性能进行进一步评估,通过测定检测不同降钙素原浓度的样本,最终确定试剂的最低检出量为0.2ng/ml;利用C-反应蛋白、降钙素、类风湿因子阳性样本评价试剂的特异性,发现本试剂与10mg/m1的C-反应蛋白、6.4mg/m1的降钙素及500IU/m1的类风湿因子均无交叉反应,特异性良好；血清血浆样本检测结果一致性考核方面,将PCT标本分别处理成血清、肝素抗凝血浆、EDTA抗凝血浆样本,确定血清、血浆检测结果是否一致,最终通过实验发现试剂在这两种样本的检测性能上具有一致性；本试剂经稳定性评价发现其在37℃条件下放置21天不会对试剂性能造成影响：精密性评价是平行测定0.5ng/ml、2ng/ml及10ng/ml参考品各十次,观察其显色程度,通过平行实验发现,本试剂精密性良好；最后将本试剂与定量试剂盒进行比较,取临床定量检测样本45例,用本试剂进行半定量检测,将二者的结果进行对比,以评价试剂半定量的准确性,通过实验发现,二者的符合率为88.9%。
Immunolabelling technologies are method for qualitative and quantitative determ ination of the body fluid of the hapten, antigen or antibody. The antigen antibody reac tion was labeled antibody or antigen by using of fluorescein, radioactive isotope, enzy me, ferritin, colloidal gold and chemical (or biological) luminescence agent as tracer. These methods also with the help of fluorescence microscopy, X-ray measuring instru ment, ELISA test, electron microscopy and luminescence immunoassay instrument pr ecision application of various instruments, liquid phase and solid phase immunoassay m-ethod.
Time-resolved fluorescence immunoassay (Timed-resolved fluoroimmunoassay, TRFIA) is an advanced sensitive quantitative immunoassay technology. The technolo gy is with lanthanide ions as a marker to mark the antigen or antibody. Because of Ian thanide ions have unique fluorescence properties, the technology can achieve high sig nal-to-noise ratio, thus has high sensitivity, and has the advantages of simple preparati on, markers of storage time, free of radioactive pollution, detection reproducibility, sh ort operating process, the standard curve range is wide, not affected by sample natural fluorescence interference and the application range is very extensive. Time-resolved fluorescence immunoassay compared with enzyme immunoassay, radioimmunoassay has higher clinical application value. Clinical detection of the technology is suitable f or all kinds of antigen and antibody, has been widely used in the market.
Testosterone is a hormone, which detection is one important part of clinically six h-ormone detection. Clinically used in testosterone detection reagent mainly are the c he-miluminescence detection reagent which imported and time-resolved testosterone assay kit from testosterone production company PE currently. Testosterone detection reagent and the development of domestic become the focus of our attention. Testoster one as the steroid hormone, is a small molecular hapten molecules in serum testostero ne,98%are present in combination with hormone binding protein, which has brought the difficulty and challenge these particularities to testosterone ELISA R&D work. Therefore, in the development process of testosterone reagent, we will introduce the i dea of heterologous bridge with the development process, structural analogues of test osterone androstene two ketone as the carrier, in its C3introduction containing five m ethylene arm, on the basis of the structure of synthetic NHS active ester, and BSA co-upling by Eu markers thus as a tracer in the reaction system. The indirect competition method was developed for determination of the reagent, serum testosterone levels in plasma.
Colloidal gold immune chromatography is a new technology based on ELISA, la-tex agglutination test, monoclonal antibodies and immune colloidal gold labeling tec-hnique, which is marked by colloidal gold, to amplify the response using the antigen antibody reaction specificity. It can be judged by direct observation. This technology has the advantages of simple, rapid, accurate and no pollution. Development of variou-s diagnosis field in clinical medicine detection, hormone detection, detection of food safety, drug residue rapid detection and drug fast [1]. With the development of instant test (POCT) in-depth, colloidal gold reagent has become the main field for the develo pment of POCT at present because of its fast, convenient, with easy to carry, easy to i-ndepth community and remote and a mountainous area, so the requirement of colloid-al gold reagent for detecting acute onset, the harm of diseases related became urgenc-y.
This article is composed by three parts.
In the first part, four kinds of testosterone and testosterone analogues chemical derivatives and conjugates prepared. In the produce, with testosterone or testosterone analogues-androstene as raw material, then introduced O-(carboxymethyl) hydroxylamine hydrochloride (O-(carboxymethyl) hydroxylamine hemi-hydrochloride) or aminooxy acetic acid hydrobromide (6-Aminooxy-hexanoic acid, hydrobromide) as the arm structure. In the next step, the active ester was synthesized as following:in the NHS and DCC conditions, stirring overnight, the oxime synthesis of NHS corresponding to the active ester structure, use flash's chromatography to remove unreacted components. Then took active ester and BSA scale with the molar ratio of15:1coupling reaction at room temperature, stirring overnight, the next day to molecular sieve purification, and four types of conjugates concentrations were detected by BCA method. At last, concentrations of testosterone conjugates I, II, III and IV were0.8mg/ml、0.9mg/ml、0.9mg/ml and0.8mg/ml.
In the second part, we described the development of testosterone time-resolved fluorescence immunoassay reagent. First, steroid-free serum was prepared with kaolin and activated carbon, and calibrator was prepared with the steroid-free serum just as following:A(0nmol/L)、B (1.0nmol/L)、C(3.2nmol/L)、D(6.8nmol/L)、 E(14nmol/L)and F(51.2nmol/L). The next step is the labelling of testosterone conjugates. Mixed each conjugate1mg with0.5mg Eu3+and incubated for18h at room temperature. The product was purified and detect the testosterone concentration by BCA assay, then the Eu standard solution was added to the purified product and preserved under-20℃.The concentration of testosterone tracers were132μg/ml、135μg/ml、138μg/ml和140μg/ml separately. Step three is the definition of the optimum coating concentration of goat anti-mouse IgG. Detected and compared the fluorescence value of testosterone calibrator A in different coating concentration separately, the optimal goat anti-mouse IgG coating concentration is6μg/ml. Step four is the comparison of four testosterone tracers. Detected the ED50of each testosterone tracer, and chose tracer IV with the lowest ED50as the optimal testosterone tracer. Step five is the definition of sample quantity. Detected the fluorescence value of testosterone calibrator A (Bmax) and the fluorescence value of testosterone calibrator B under different sample quantity separately, calculated the B/Bmax ratio, chose25μl with the lower B/Bmax ratio and the easier operation procedure as the optimal sample amount. Step six is the definition of optimal concentration of testosterone monoclonal antibody and testosterone tracer. The srandard of determination is sensitivity and fluorescence value of testosterone calibrator A, finally the optimal concentration of testosterone monoclonal antibody is6ng/ml, the optimal concentration of testosterone tracer is1μg/ml. In human serum,99%of testosterone is bind with SHBG, so the blocking agent plays important role in the exact detection of testosterone. Step seven is the determination of optimum concentration of blocking agent. Detected the fluorescence level of testosterone calibrator, calculated and compared the B/Bmax of each calibrator in different concentration of blocking agent, evaluated the sensitivity and influence of blocking agent concentration on the reaction system, determined the optimal concentration of blocking agent was30μg/ml. Clarifyed the optimum reaction time in step eight, detected the fluorescence ratio with calibrator A, B, C, D, E,F and Btotal in different reaction time, eventually optimal reaction time is90minutes.Overall evaluation of the reagent was carried out after all the parameters were determined. Analytical sensitivity is0.15nmol/L; The recovery rate of dilution is between98.8%and101.4%, there is a linear relationship between the dilution and concentration. Then analysis the specificity of the reagent, the cross-reactivity of progesterone was0.14%, the cross-reactivity of DHT was0.38%, the cross-reactivity of estradiol was0.07%, the cross-reactivity of17-hydroxyl progesterone was0.02%, and the cross-reactivity of estriol was0.09%, and the cross-reactivity of Danazol was0.12%. In the aspect of precision analysis, the coefficient of variation (CVs) was2.5-6.6%(intraassay)and3.6%～7.8%(interassay). In terms of the evaluation of linear, linear formula is Y=1.453-2.193X, the correlation coefficient r=0.99, R2=1.000,F=7303.334,P<0.001, shows that the linearity of this competition system is perfect; Compared with PE reagent kit,84samples were detected, correlation formula was Y=0.991X-0.331, the correlation coefficient was0.988, R2=0.977, F=3407.808, P<0.001, the result reflected that this reagent can meet the requirements of clinical detection very well.
The third part of the thesis mainly introduces the development process of procalcitonin colloidal gold immunochromatography semi-quantitative detection.In the reagent development process, the NC membrane was screened at first, HF135was determined by the comparison of capillary flow, sensitivity, response time and the background; the second step is the definition of the colloidal gold combined pad material, The selection of colloidal gold candidate materials were compared from glass fiber materials, the nonspecific adsorption, colloidal gold constant release, colloidal gold dissolution and release rate, and hemolysis prevention. The final determination is GFCP103000produced by Millipore after a series of experiments; the third step is the screening of raw materials by TRFIA and colloidal gold method. Evaluated29combinations of antibodies successively, contain pairing specificity, sensitivity, color discrimination, and so on. Ultimately,16B5of Hytest was determined as coating material, MJG03of Hangzhou Qi Tai was determined as labeling material. The fourth step is definition of the optimal labeling condition of MJG03, the optimum labeling conditions of MJG03is15μl0.1M K2CO3per milliliter of colloidal gold, and1.5mg MJG03every100ml colloidal gold. The fifth step is to determine the best coating condition of16B5, lOmM PBS (pH7.2) was chosen as the coating buffer by setting different packages. Detection in different coating conditions reagent sensitivity, specificity, T line color degree conditions, choose the best performance of T line. The sixth step is the determination of the concentration of16B5and gold-labeled MJG03, ultimately the concentration of gold-labeled MJG03is OD535=40, the spraying quantity is0.5μl/mm, the concentration of16B5is2.5mg/ml, the spraying quantity is0.12μl/mm, the optimal concentration of goat anti mouse IgG is lmg/ml, the spraying quantity is0.12μl/mm. The seventh step is the establishment of standard, the positive samples of0.5ng/ml,2ng/ml and lOng/ml were measured for20times separately, and then took the average chromaticity as color standard. The evaluation of reagent carried out after related parameters were determined, the sensitivity of reagent is0.2ng/ml, and the reagent did not react with C-reactive protein(10mg/ml), calcitonin(6.4mg/ml), and rheumatoid factor(500IU/ml). The consistency of plasma and serum is perfect. The performance of reagent did not changed under the condition of37℃for21days. The precision of reagent was evaluated by samples with the concentration of0.5ng/ml,2ng/ml and10ng/ml for ten times, and this experiment showed that precision of reagent was perfect. Finally, the reagent was compared with other quantitative reagent, the correlation coefficient is88.9%.
引文
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[17]HOLGATE C S, JACKSON P, COWEN P N, et al. Immunogold silver staining: new method of immunostaining with enhanced sensitivity[J]. J Histochem Cytochem,1983,31(7):938-944.
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