CN112946254B - 一种胶乳增强竞争免疫比浊检测方法及试剂盒 - Google Patents
一种胶乳增强竞争免疫比浊检测方法及试剂盒 Download PDFInfo
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- CN112946254B CN112946254B CN202110064759.6A CN202110064759A CN112946254B CN 112946254 B CN112946254 B CN 112946254B CN 202110064759 A CN202110064759 A CN 202110064759A CN 112946254 B CN112946254 B CN 112946254B
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Landscapes
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Abstract
本发明公开了一种用于改善胶乳增强竞争免疫比浊分析灵敏度的方法以及基于该方法的检测试剂盒。本发明利用生物素对抗体的多重标记和生物素‑亲合素之间的结合反应,可以使用微量的BLAAA建立胶乳增强竞争免疫比浊分析方法,显著改善检测敏感性。
Description
技术领域
本发明属于生化检测领域,具体涉及一种胶乳增强竞争免疫比浊检测方法及试剂盒。
背景技术
胶乳增强免疫比浊检测(Latex enhanced turbidimetric immunoassay,LETIA)是一种经典的免疫检测方法,它将抗体或抗原偶联于纳米尺寸的胶乳微球表面,利用抗体-抗原间高度特异性的结合,使胶乳微球表面的抗体或抗原与标本所含的待测物反应而形成微球聚集体,进而使免疫反应体系在特定波长下的吸光度发生变化,通过测定该变化计算标本中待测物的浓度。迄今,LETIA在临床医学中已得到广泛应用,涉及肿瘤、风湿、肝功能、肾功能和药物浓度监测等诸多领域。然而,尽管LETIA已广泛应用于常规临床分析,但由于该方法较低的分析灵敏度,以LETIA准确定量标本中含量极低的待测物浓度,仍然是一个挑战。
胶乳增强竞争免疫比浊检测是LETIA的一种实施方式,其通过标本所含待测物与检测体系固定量的待测物竞争结合限量的抗待测物抗体实现对待测物的定量,常用于小分子待测物的检测,如:地高辛、他克莫司(Tacrolimus)、环孢素、甲胺喋呤、伊马替尼、黄曲霉素、克伦特罗和苏丹红,等。
被本领域技术人员所熟知的用于小分子待测物检测的增强竞争免疫比浊检测通常采用待测物小分子偶联微球直接法和抗体偶联微球直接法两种模式,其原理分别示于图1-B及图1-C。
在待测物小分子偶联微球直接法模式中(图1-B),标本所含待测物小分子与偶联于微球表面的待测物小分子竞争结合限量的抗待测物抗体,当标本中不含待测物时,抗体可通过其二个结合位点桥联二个不同的微球,从而引发凝集;当标本中含有待测物时,抗体结合位点被待测物占据,胶乳微球的凝集受到抑制。该模式下,抗体的两个结合位点均与同一胶乳微球表面的二个待测物分子结合的那部分抗体,将失去桥联两个胶乳微球的能力,从而无法引发凝集。尽管经过充分的工艺优化,本发明人采用此模式建立的他克莫司LETIA,其可获得的他克莫司最高灵敏度仅约6.9ng/mL。
抗体偶联微球直接法(图1-C)需要先将小分子与蛋白质或其它载体偶联,制备载体-小分子复合物,使单一载体分子上含有多个小分子;由于载体-小分子复合物和抗体-胶乳微球的多价性,在适当的条件下载体-小分子复合物与抗体-胶乳微球很容易引发凝集,而标本中的小分子待测物通过占据胶乳微球表面的抗体结合位点而抑制这种凝集反应,根据凝集反应的被抑制程度,可定量标本中的小分子待测物浓度。本发明人采用此模式研制了人全血他克莫司胶乳增强竞争免疫比浊检测试剂(CFDA注册证号:20192400305),尽管其具有优良的检测准确性,可获得的他克莫司LOD(检测下限,Limit of Detection)仅约1.0ng/mL。1.0ng/mL的LOD对于大多数他克莫司临床监测已足够灵敏,但对于联合免疫抑制用药的器官移植患者(A comparative,randomized trial of concentration-controlledsirolimus combined with reduced-dose tacrolimus or standard-dose tacrolimusin renal allograft recipients.Transplantation Proceedings,2013,45,2133-2140)和某些使用低剂量他克莫司治疗的自身免疫性疾病患者(Changes in the blood level,efficacy,and safety of tacrolimus in pregnancy and the lactation period inpatients with systemic lupus erythematosus.Lupus.2018,27:2245-2252),进一步改善其检测灵敏度将有助于更准确地检测这些患者血液中的低浓度药物。
因此,寻找可以有效改善胶乳增强竞争免疫比浊检测灵敏度的方法,将有助于更准确地检测标本中低含量的小分子待测物的浓度,拓展胶乳增强竞争免疫比浊检测的待测物范围。
发明内容
为解决现有胶乳增强竞争免疫比浊检测灵敏度低、难以准确检测低浓度小分子待测物的技术问题,本发明提供一种新的胶乳增强竞争免疫比浊检测模式(如图1-A所示)以及基于该检测方法的试剂盒。该模式下,由于生物素对抗待测物抗体的多重标记,生物素标记的抗待测物抗体(Biotin labeled anti-analyte antibodies,BLAAA)与胶乳微球表面的待测物结合可在所述胶乳微球表面引入大量的生物素,即使使用少量的BLAAA,也可在四价亲合素的存在下引发有效的凝集反应,因此,本模式可以使用微量的BLAAA建立胶乳增强竞争免疫比浊检测方法,显著改善检测灵敏度。
本发明提供了一种胶乳增强竞争免疫比浊检测方法,其包含如下步骤:
(1)将生物素标记的抗待测物抗体(Biotin labeled anti-analyte antibodies,BLAAA)与校准品或标本混匀,孵育,得混合液1;
(2)将步骤(1)中所述混合液1与亲合素和待测物偶联的胶乳微球混匀,并检测初始吸光度A1,孵育后检测吸光度A2,计算吸光度增值ΔA,ΔA=A2-A1;
(3)以校准品的吸光度增值ΔA对其所含待测物浓度建立校准曲线,通过比对标本的吸光度增值ΔA实现对所述标本中待测物浓度的定量检测。
或,
(1)将生物素标记的抗待测物抗体(BLAAA)与校准品或标本混匀,孵育,得混合液1;
(2)将步骤(1)中所述混合液1与待测物偶联的胶乳微球混匀,孵育,得混合液2;
(3)将步骤(2)中所述混合液2与亲合素混匀,并检测初始吸光度A1,孵育后检测吸光度A2,计算吸光度增值ΔA,ΔA=A2-A1;
(4)以校准品的吸光度增值ΔA对其所含待测物浓度建立校准曲线,通过比对标本的吸光度增值ΔA实现对所述标本中待测物浓度的定量检测。
本发明中,所述抗待测物抗体指能与待测物发生特异性免疫结合反应、具有抗体结构特征的蛋白质。
本发明中,所述待测物指胶乳增强竞争免疫比浊检测可检测的人体、动物体、植物体以及自然界非生物体所含的各种小分子物质,如:地高辛、他克莫司(Tacrolimus)、环孢素、氯氮平、阿立哌唑、奥氮平、***、喹硫平、利培酮、阿米替林、度洛西汀、去甲替林、文拉法辛、西酞普兰、丙咪嗪、舍曲林、卡马西平、奥卡西平、丙戊酸、苯妥英、拉莫三嗪、左乙拉西坦、托吡酯、黄曲霉素、两性霉素B、伏立康唑、泊沙康唑、伊曲康唑、替考拉宁、万古霉素、利奈唑胺、阿米卡星、多粘菌素B、替加环素、氯霉素、三聚氰胺、伏马毒素、黄曲霉素M1、黄曲霉素B1、克伦特罗或沙丁胺醇。
本发明中,所述生物素标记的抗待测物抗体可采用生物素标记试剂,按本领域常用的生物素标记方法制得。所述的生物素标记试剂包括但不限于与蛋白质氨基反应的各种臂长的生物素琥珀酰亚胺酯,与蛋白质巯基反应的各种臂长的生物素马来酰亚胺衍生物,与蛋白质羧基反应的各种臂长的生物素氨基化衍生物。本领域技术人员可采用商品试剂盒(如:Biotin Quantitation Kit;Thermo-fisher,US)按HABA-Avidin法确定生物素对抗待测物抗体的标记率。
所述BLAAA中,所述生物素与所述抗待测物抗体的摩尔比优选为5-25:1,更优选为10-20:1,最优选为12-17:1。
所述BLAAA以pH缓冲液的形式贮存和使用。所述BLAAA的浓度一般为1-1000ng/mL,优选为5-200ng/mL。
本发明中,所述亲合素指含有四个生物素结合位点、能与所述生物素分子高亲合结合的一组蛋白,如亲合素(Avidin)、中和亲合素(Neutravidin)、链霉亲合素(Streptavidin)或含上述各种亲合素的聚合物。所述亲合素以pH缓冲液的形式贮存和使用,所述亲合素的浓度优选为0.5-500μg/mL,更优选为2-100μg/mL。
本发明中,所述待测物偶联的胶乳微球可按本领域常规方法,采用化学偶联或物理吸附方式制得。如果所述待测物含有可直接与所述胶乳微球的表面基团发生偶联反应的活性官能团,可依据所述胶乳微球和所述待测物所含的官能团选择相应的化学方法实施偶联;也可以先将所述待测物偶联于蛋白,得到蛋白-待测物复合物,再通过所述蛋白所含的反应官能团与所述胶乳微球的表面官能团进行化学偶联,将所述蛋白-待测物偶联于所述胶乳微球表面,完成所述待测物对所述胶乳微球的间接偶联。
所述待测物偶联的胶乳微球中,所述胶乳微球可选择本领域常用的胶乳微球,例如,表面含羧基的聚苯乙烯胶乳微球。所述表面含羧基的聚苯乙烯胶乳微球的平均粒径优选为50-500nm,更优选为100-400nm。所述表面含羧基的聚苯乙烯胶乳微球的表面羧基密度为0.02-0.40mmol/g,优选为0.06-0.20mmol/g。
所述待测物偶联的胶乳微球以pH缓冲液的形式使用。所述待测物偶联的胶乳微球的浓度一般为0.01%-1%,优选为0.05%-0.5%。
本发明中,所述pH缓冲液中的缓冲剂可按本领域常规选择下述任一试剂与其共轭酸或其共轭碱组成的缓冲对:三羟甲基氨基甲烷(Tris)、N-2-羟乙基哌嗪-N-3丙磺酸(HEPPS)、3-(N-***代)丙烷磺酸(MOPS)、三乙醇胺(TRA)、二乙醇胺(DEA)、2-甲基-2-氨基-1丙醇(AMP),N-双(2-羟乙基)-2-氨基乙磺酸(BESN)、(环己胺)-1-丙磺酸(CAPS)、(环己胺)-1-乙磺酸(CHES)、(N-***啉)乙磺酸(MES)、哌嗪-N,N-双(2-乙磺酸)(PIPES)、哌嗪-N,N-双(2-羟基乙烷磺酸)(POPSO)、三(羟甲基)甲基-3-氨基丙烷磺酸(TAPSN)和三(羟甲基)甲基-2-氨基磺酸(TESN)中的一种或多种。所述pH缓冲液的pH优选为4.5-9.5,更优选为6.0-8.0。所述缓冲剂的浓度可按常规选择,一般优选为10-500mM。
在所述待测物偶联的胶乳微球或含有所述BLAAA的pH缓冲液中添加盐类化合物可增加溶液的离子强度,有利于所述微球和所述BLAAA的稳定贮存。所述离子强度主要来自于碱金属盐溶液,包括但不限于NaCl、KCl、Na2HPO4、NaH2PO4、K2HPO4、KH2PO4、Na2CO3、NaHCO3和K2CO3。所述碱金属盐的浓度优选为0.01-2.00mol/L,更优选为0.05-1.0mol/L。
所述pH缓冲液中还可包含促凝剂、防腐剂、表面活性剂和封闭剂中的一种或多种。
其中,所述促凝剂可加快待测物与抗待测物抗体之间以及亲合素与生物素之间的反应。所述促凝剂的种类和使用浓度可按本领域常规选择。在本发明中,所述促凝剂优选为分子量大于3000道尔顿的聚乙二醇(PEG),更优选为分子量6000-30000道尔顿的PEG。所述促凝剂的浓度通常为0.1-10wt%。
其中,所述防腐剂可按本领域常规选择,如选自Proclin300、叠氮钠、凯松和庆大霉素。
其中,所述表面活性剂可按本领域常规选择。在本发明中,所述优选为非离子型表面活性剂,其包括但不仅限于脱水山梨醇聚氧乙烯醚月桂酸酯类表面活性剂、失水山梨醇聚氧乙烯醚油酸酯类表面活性剂和辛基酚聚氧乙烯醚类表面活性剂;所述表面活性剂的浓度一般为0.01-1wt%。
其中,所述封闭剂可按本领域常规选择,如Blockmaster DB1130和牛血清蛋白(BSA)。
本发明还提供了一种胶乳增强竞争免疫比浊检测试剂盒,其包括:
(1)试剂1(R1),所述R1为含有BLAAA的pH缓冲液,所述BLAAA为生物素标记的抗待测物抗体;
(2)试剂2(R2),所述R2为含有亲合素和待测物偶联的胶乳微球的pH缓冲液;
(3)校准品。
或,
(1)试剂1(R1),所述R1为含有BLAAA的pH缓冲液,所述BLAAA为生物素标记的抗待测物抗体;
(2)试剂2(R2),所述R2为含有待测物偶联的胶乳微球的pH缓冲液;
(3)试剂3(R3),所述R3为含有亲合素的pH缓冲液;
(4)校准品。
所述校准品为含有已知浓度的所述待测物的标准溶液或冻干品,用于建立校准曲线,实现对所述待测物的定量检测。
为简化试剂盒的组成和便于试剂盒的使用,优选地,所述胶乳增强竞争免疫比浊检测试剂盒仅包括:含有BLAAA的R1、含有亲合素和待测物偶联的胶乳微球的R2和含有已知浓度所述待测物的校准品。
优选地,所述胶乳增强竞争免疫比浊检测试剂盒的各组分同前述。
本发明所有技术方案中,若非特别注明,所述浓度均指使用前(混合前)的浓度,所述百分比(%)均为质量/体积比(g/100mL)。在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所有技术方案中,若非特别注明,本发明所用试剂和原料均市售可得。
本发明的积极效果在于:本发明提供的胶乳增强竞争免疫比浊检测方法及试剂盒可显著改善小分子待测物的检测灵敏度。基于检测灵敏度的改善,本方法可用于准确检测标本中低含量的小分子待测物的浓度,在一定程度上克服胶乳增强竞争免疫比浊检测灵敏度较低的局限,从而使更多的低浓度待测物得以实现准确检测,扩大胶乳增强竞争免疫比浊检测的待测物范围。
附图说明
图1为胶乳增强竞争免疫比浊检测的三种模式;其中,图1-A为本发明提出的胶乳增强竞争免疫比浊检测模式,图1-B为待测物偶联微球直接法,图1-C为抗体偶联微球直接法。
图2为BLAAA用量为10ng/测试时,6个校准品的凝集进展曲线。总反应体积为210μl,其中标本10μl,R1 100μl,R2 100μl。
图3为使用不同BLAAA用量时他克莫司LETIA的校准曲线。
图4为他克莫司胶乳增强竞争免疫比浊检测的功能灵敏度。
图5为用胶乳增强竞争免疫比浊检测、ABBOTT CMIA和LC-MS/MS测定119例患者全血他克莫司浓度的比较。Bland-Altman图中的实线表示两次测定的平均差值,虚线表示平均差值的95%CI。图5A和图5C:Passing and Bablok回归曲线;图5B和图5D:Bland-Altman偏差。
具体实施方式
以下以他克莫司的检测为例,阐述本发明的实施方法及其应用效果。该具体实施例用于说明本发明,但本发明范围并不受实施例内容所限制。下列实施例中未注明具体条件的实验方法,可按照常规方法和条件,或按照商品试剂盒说明书选择。
以下实验所用材料中,羧基化胶乳微球(P0323)及封闭试剂Blockmaster DB1130来自JSR(日本);抗他克莫司单克隆抗体、他克莫司-BSA复合物和校准品为自制(Feng-BoWu,Yun-Yun Yang,Xue-Bin Wang,Zhuo Wang,Wen-Wen Zhang,Zheng-Yue Liu,Yun-QiQian.A sample processing method for immunoassay of whole bloodtacrolimus.Analytical Biochemistry.2019,576,13–19);他克莫司沉淀试剂来自ABBOTTTacrolimus CMIA(Chemiluminescent magnetic immunoassay)试剂盒;生物素-抗体偶联率使用Biotin Quantitation Kit(Thermo-fisher,US)测定;其余试剂均购自Sigma公司。所有免疫比浊检测均在日立7080生化分析仪上运行。采用的质谱检测***为SCIEX TripleQuad 4500MD-JasperTM(美国);ABBOTT Tacrolimus CMIA(试剂批号:93006M800)在Architect i1000运行;使用Nanotrac Wave II纳米颗粒分析仪(美国)监测他克莫司-胶乳偶联过程;使用日立CR21N高速冷冻离心机(日本)对胶乳进行清洗和缓冲液交换;用于分散胶乳的ATS-e型超声细胞破碎器购于ATS公司(中国)。
实施例1基于检测模式A的他克莫司LETIA
1.1抗他克莫司抗体的生物素化
按文献(Feng-Bo Wu,Shi-Quan Han,Chao Zhang,and You-Feng He.Synthesisof a highly fluorescentβ-diketone-europium chelate and its utility in Time-resolved fluoroimmunoassay of serum total thyroxine.Anal.Chem.,2002,74,5882-5889;Feng-Bo Wu,Hai-Qiao Ouyan,Xiao-Yan Tang and Zhen-Xian Zhou.Double-antigen sandwich time-resolved immunofluorometric assay for the detection ofanti-hepatitis C virus total antibodies with improved specificity andsensitivity.Journal of Medical Microbiology.2008,57,947–953)所述方法制备生物素标记的抗他克莫司抗体,即:将抗他克莫司单克隆抗体与生物素化-epsilon-氨基己酸-N-羟基丁二酰亚胺反应,经透析去除游离生物素试剂。生物素对抗体的偶联率使用生物素定量试剂盒(Biotin quantitation kit,Thermo-fisher)通过HABA-Avidin法测定,生物素:抗体分子=16:1。
1.2他克莫司偶联的胶乳微球的制备
将50mg羧基化胶乳微球悬浮于5mL MES缓冲液(20mM,含2%NaCl,pH6.2)中,加入5mg EDC和50mg N-羟基磺基琥珀酰亚胺钠(NHSS),室温搅拌下反应45min。12000×g离心15min,活化的胶乳微球在5mL偶联缓冲液(100mM Hepes,pH7.5,含2%NaCl)中超声重悬,与含2.5mg他克莫司-BSA的250μl生理盐水混合,于室温搅拌下孵育过夜。12000×g离心胶乳微球15min,重悬于10mL洗涤pH缓冲液(20mM Hepes,pH8.5,含0.5%NaCl,0.05%Tween-20,0.05%NaN3)中,以3.5mm变幅杆在35%最高频率下超声30秒。重复两次上述重悬-洗涤-超声步骤,以确保去除游离的他克莫司-BSA偶联物;最后,将微球重悬于pH缓冲液中得R2,使制备的R2在546nm波长下的吸光度为2.6±0.6A(UV-1900型岛津紫外-可见分光光度计)。
1.3他克莫司LETIA测定
经优化的他克莫司LETIA试剂:R1为含有0.9%NaCl、100±20ng/mL生物素标记的抗他克莫司抗体、3mg/mL EDTA-2K、3%PEG-8000、2%蔗糖和0.5%BSA的20mM MES(pH 6.1)缓冲液;R2为含2%NaCl、5%蔗糖、0.1%Tween-20、0.5%Blockmaster DB1130、8±2μg/mL链霉亲合素(SA)和0.1±0.05%他克莫司偶联的胶乳微球的50mM Hepes(pH 7.5)缓冲液。
在LETIA测定标本之前,先使用沉淀试剂从EDTA全血中提取他克莫司:将300μl全血标本或校准品转移到含300μl沉淀试剂的1.5mL EP管中,强力涡旋10秒钟后于15000×g离心5分钟,将他克莫司上清倒入标本杯,转移到生化分析仪测定他克莫司。
检测前,按下表数据设置设备参数:
按下表数据设置检测参数:
图2为BLAAA用量为10ng/测试时,6个不同他克莫司浓度的校准品的凝集进展曲线;总反应体积为210μl,其中校准品10μl,R1 100μl,R2 100μl。
图3为使用不同BLAAA用量时他克莫司LETIA的校准曲线。
当BLAAA用量为5ng/测试、10ng/测试时,他克莫司LETIA的LOD分别为0.12ng/mL、0.27ng/mL,定义为平行测定24次浓度为零的校准品所得的平均ΔA减2SDs在校准曲线上所对应的他克莫司浓度(图3,曲线4、5)。
实施例2基于检测模式A的他克莫司LETIA、CMIA和LC-MS/MS的临床标本测定值比较
用于对比研究的119例全血EDTA标本中,81例来自肾移植患者,38例来自肝移植患者。分别以实施例1所述的LETIA、雅培CMIA和LC-MS/MS测定这些标本中的他克莫司浓度;其中,ABBOTT Tacrolimus CMIA在Architect I1000分析仪上严格按照试剂盒说明书进行,LC-MS/MS按下述方法实施。
LC-MS/MS:采用SCIEX Triple Quad 4500MD-JasperTM***(AB SCIEX)进行LC-MS/MS分析。将标本和校准品各200μl加入2ml EP管中,再加入1000μl含5ng安定(diazepam,内标)的乙腈,混合物漩涡1min,13000×g离心8分钟。取600μl上清至2mL EP管中,与600μl纯水混合,涡旋10秒后再次13000×g离心8分钟,将上清转移到自动采样瓶中,注射10μl上清标本于LC-MS/MS***中。他克莫司在Hypersil Gold C-18柱(3μm,2.1×100mm)上分离,梯度流动相由溶剂A(乙腈)和溶剂B(纯水)组成。所有程序的流速均为0.4mL/min。柱温45℃。初始2分钟的流动相为10%A和90%B的混合物,随后将溶剂A增加到90%,保持3分钟。最后,在5.1分钟时将溶剂A降至10%,保持2分钟;然后注射下一个标本。质谱仪设置:喷雾电压5.5KV,幕气:30psi,碰撞气体(CAD):9,源GS1:45psi,源GS2:45psi,落差温度400℃。他克莫司和内标安定监测的MRM m/z比值分别为m/z 821.5→768.5/786.6和285.0→222.1。采用TRIPLE QUADTM 4500MD***对离子色谱峰进行集成和定量。
以MedCalc-15.2.2对LETIA、LC-MS/MS和ABBOTT CMIA检测的标本他克莫司浓度作线性回归和Bland-Altman分析(图5),线性回归方程分别为YLETIA=0.998XLC-MS/MS+0.51(R2=0.977)和YLETIA=1.01XCMIA+0.13(R2=0.982),LETIA对CMIA和LC-MS/MS的平均偏离分别为-0.2ng/mL和-0.5ng/mL。虽然没有检测LETIA与他克莫司代谢物的交叉反应性,由于LC-MS/MS的高度特异性以及LETIA与LC-MS/MS测定值的高度一致性,可以推断,本实施例建立的他克莫司LETIA对他克莫司代谢物的交叉反应性不影响测定。
图5显示了LETIA、ABBOTT CMIA和LC-MS/MS测定119例患者全血他克莫司浓度的方法间的一致性。Bland-Altman图中实线表示两次测定的平均差值,虚线表示平均差值的95%CI。A、C:Passing and Bablok回归曲线;B、D:Bland-Altman偏差。
下述实施例3、4和5的他克莫司LETIA分析性能评估实验,参照实施例1中所述方法进行。
实施例3基于检测模式A的他克莫司LETIA的功能灵敏度及精密性
将他克莫司浓度为12.8ng/mL、10.6ng/mL和8ng/mL的3份血样倍比稀释至他克莫司浓度小于0.15ng/mL,对每个标本进行重复检测,每天两次,连续测定10天,计算每个浓度的平均浓度和%CV,根据所得数据以Average multiple curves fitting拟合(图4),他克莫司的功能灵敏度定义为变异系数(CV,%)为20%时所对应的他克莫司浓度,为0.59ng/mL。图4示意了他克莫司原始浓度为12.8ng/mL(菱形)、10.6ng/mL(正方形)和8ng/mL(三角形)的3份血样倍比稀释后的各他克莫司浓度与其所对应的实验间CV的数量关系。
同一次测定中,20次平行测定浓度分别为5.1、13.9和20.2ng/mL的3份他克莫司全血标本,实验内CV分别为3.6%、3.9%和4.6%。以1天为间隔,重复20天测定上述3份标本,实验间CV分别为3.3%、5.2%和5.1%。
实验内CV:同一次测定中,平行多孔测定某标本所得浓度值的CV。
实验间CV:不同次测定中,测定某标本所得浓度值的CV。
实施例4基于检测模式A的他克莫司LETIA回收率
以不含他克莫司的校准品(零浓度校准品)连续稀释高浓度他克莫司标本(29.8ng/mL),根据测定值和理论值计算回收率,结果如表1。
表1他克莫司LETIA的稀释线性
#:计算值=测定值乘以稀释系数
&:回收率(%)=(计算浓度/稀释前浓度)×100
实施例5基于检测模式A的他克莫司LETIA抗干扰实验
以下述小分子测试本发明基于检测模式A的他克莫司LETIA的交叉反应。在零浓度校准品中添加西罗莫司和依维莫斯使其终浓度均为100ng/mL,在零浓度校准品中添加环孢霉素使其浓度为2000ng/mL,在零浓度校准品中添加霉酚酸、阿米卡星、两性霉素B、酰胺、卡马西平、氯霉素、呋喃苯胺酸、青霉素、卡那霉素、万古霉素和替考拉宁,使它们的浓度均为100μg/mL,以他克莫司LETIA检测所有上述制备的标本,所得的他克莫司平均浓度(n=3)均小于功能灵敏度(0.59ng/mL)。
分别在胆固醇含量为339mg/dL、甘油三酯含量为727mg/dL和胆红素含量为30.0mg/dL的临床标本中加入他克莫司使其浓度为10ng/mL,以他克莫司LETIA检测他克莫司的浓度,测定值误差均小于15%。
由于没有商业化的他克莫司代谢物,未检测与他克莫司代谢物的交叉反应程度。也未检测蛋白质来源的干扰,如类风湿因子(RF)和嗜异性抗体,因为这类干扰物质已经在测试前的标本预处理步骤中沉淀并去除。
以上实验结果表明,BLAAA用量为5ng/测试、10ng/测试时,本发明所述的基于检测模式A建立的他克莫司LETIA可分别获得0.12ng/mL、0.27ng/mL的LOD;他克莫司LETIA与LC-MS/MS和ABBOTT CMIA对标本测定值的高度一致证明了本发明LETIA模式的测量准确性。基于这些结果,本发明所述的胶乳增强竞争免疫比浊检测方法不仅可用于开发高灵敏的他克莫司LETIA,还可望成为一种通用的方法用于其它低浓度小分子的检测。基于检测灵敏度的改善,本方法可扩大传统LETIA技术的可测待测物的范围。
Claims (10)
1.一种胶乳增强竞争免疫比浊检测方法,其包含如下步骤:
(1)将生物素标记的抗待测物抗体(Biotin labeled anti-analyte antibodies,BLAAA)与校准品或标本混匀,孵育,得混合液1;
(2)将步骤(1)中所述混合液1、待测物偶联的胶乳微球和亲合素混匀,并检测初始吸光度A1,孵育后检测吸光度A2,计算吸光度增值∆A,∆A =A2-A1;
(3)以校准品的吸光度增值∆A对其所含待测物浓度建立校准曲线,通过比对标本的吸光度增值∆A实现对所述标本中待测物浓度的定量检测;
或,
(1)将生物素标记的抗待测物抗体(BLAAA)与校准品或标本混匀,孵育,得混合液1;
(2)将步骤(1)中所述混合液1与待测物偶联的胶乳微球混匀,孵育,得混合液2;
(3)将步骤(2)中所述混合液2与亲合素混匀,并检测初始吸光度A1,孵育后检测吸光度A2,计算吸光度增值∆A,∆A =A2-A1;
(4)以校准品的吸光度增值∆A对其所含待测物浓度建立校准曲线,通过比对标本的吸光度增值∆A实现对所述标本中待测物浓度的定量检测;
所述待测物为他克莫司。
2.如权利要求1所述的胶乳增强竞争免疫比浊检测方法,其特征在于,所述亲合素为含有四个生物素结合位点、能与生物素高亲合性结合的一组蛋白,为亲合素、亲合素聚合物、中和亲合素、中和亲合素聚合物、链霉亲合素或链霉亲合素聚合物。
3.如权利要求1所述的胶乳增强竞争免疫比浊检测方法,其特征在于,所述BLAAA中,所述生物素与所述抗待测物抗体的摩尔比为5-25:1。
4.如权利要求3所述的胶乳增强竞争免疫比浊检测方法,其特征在于,所述生物素与所述抗待测物抗体的摩尔比为10-20:1。
5.如权利要求4所述的胶乳增强竞争免疫比浊检测方法,其特征在于,所述生物素与所述抗待测物抗体的摩尔比为12-17:1。
6.如权利要求1所述的胶乳增强竞争免疫比浊检测方法,其特征在于,所述BLAAA、所述亲合素和所述待测物偶联的胶乳微球以pH缓冲液的形式贮存和使用;
所述BLAAA的浓度为1-1000 ng/mL;
所述亲合素的浓度为0.5-500 µg/mL;
所述待测物偶联的胶乳微球的浓度为0.01%-1%。
7.如权利要求6所述的胶乳增强竞争免疫比浊检测方法,其特征在于,
所述BLAAA的浓度为5-200 ng/mL;
所述亲合素的浓度为2-100 µg/mL;
所述待测物偶联的胶乳微球的浓度为0.05%-0.5%。
8. 如权利要求1所述的胶乳增强竞争免疫比浊检测方法,其特征在于,所述胶乳微球为表面含羧基的聚苯乙烯胶乳微球;所述表面含羧基的聚苯乙烯胶乳微球的平均粒径为50-500 nm;所述表面含羧基的聚苯乙烯胶乳微球的表面羧基密度为0.02-0.40 mmol/g。
9. 如权利要求8所述的胶乳增强竞争免疫比浊检测方法,其特征在于,所述表面含羧基的聚苯乙烯胶乳微球的平均粒径为100-400 nm;所述表面含羧基的聚苯乙烯胶乳微球的表面羧基密度为0.06-0.20 mmol/g。
10.一种胶乳增强竞争免疫比浊检测试剂盒,其包括:
(1)试剂1(R1),所述R1为含有BLAAA的pH缓冲液,所述BLAAA为生物素标记的抗待测物抗体;
(2)试剂2(R2),所述R2为含有待测物偶联的胶乳微球和亲合素的pH缓冲液;
(3)校准品,所述校准品为含有已知浓度的待测物的标准溶液或冻干品;
或,
(1)试剂1(R1),所述R1为含有BLAAA的pH缓冲液,所述BLAAA为生物素标记的抗待测物抗体;
(2)试剂2(R2),所述R2为含有待测物偶联的胶乳微球的pH缓冲液;
(3)试剂3(R3),所述R3为含有亲合素的pH缓冲液;
(4)校准品,所述校准品为含有已知浓度的待测物的标准溶液或冻干品;
所述待测物为他克莫司。
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