CN109444434B - 双抗原夹心检测抗体的方法 - Google Patents
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- CN109444434B CN109444434B CN201811558639.6A CN201811558639A CN109444434B CN 109444434 B CN109444434 B CN 109444434B CN 201811558639 A CN201811558639 A CN 201811558639A CN 109444434 B CN109444434 B CN 109444434B
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Abstract
本发明涉及一种双抗原夹心检测抗体的方法,所述方法以形成固相支持物‑第一抗原Ag1‑待检测抗体‑第二抗原Ag2的形式完成检测,其中Ag2偶联有用于显示信号强度的标记物,所述方法包括以下步骤:1)将Ag1、Ag2与待检物在足以发生抗体/抗原结合反应的条件下接触,形成免疫复合物;其中以摩尔数计,Ag1的含量多于Ag2;2)洗去未结合的待检测抗体;3)加入Ag2并使其与所述免疫复合物中剩余的抗原结合位点结合;4)检测所述标记物,以指示所述待检测抗体的存在和/或含量。与现有技术相比,本发明所提供的方法既保证了检测低值灵敏度,又能解决钩状效应,降低漏检率。
Description
技术领域
本发明涉及分子生物学技术领域,具体而言,涉及一种双抗原夹心检测抗体的方法。
背景技术
抗体检测是目前病原体普筛的主要方法之一,通常采用血清学检查,免疫血清学检查具有方便、快捷和准确等优点,已经普遍应用于医疗检测和诊断领域。
从免疫学原理上来说,抗体检测一般有两种方法:间接法和夹心法。间接法的原理是血清中的抗体识别并结合包被在固定相上的抗原,去杂后,由能识别抗体的二抗结合与抗原结合在固定相上的血清抗体,形成固定相-抗原-血清中抗体-抗抗体复合物。将没有反应的多余的抗抗体洗去后,由于二抗连接有酶等标记物,能够通过添加底物产生颜色反应或发光,通过测试吸光度或者发光光子量就可以确定与血清中抗体结合的二抗的量,二抗的量与血清中的抗体含量成正比,所以可以通过检测抗抗体的量可以知道血清中特异性抗体的含量。夹心法的原理是血清中的抗体识别包被在固定相上的抗原,同时识别并结合连接有酶等标记物的抗原,产生固定相抗原-血清抗体-标记抗原夹心复合物,将多余的没有反应的物质洗掉后,标记抗原上的酶通过添加底物产生颜色反应或发光,通过测试吸光度或者发光光子量就可以检测标记抗原的含量,标记抗原的含量与血清抗体量成正比,所以可以通过检测与血清结合的标记抗体的量检测血清中特异抗体的含量。其中双抗原夹心法相对于间接法占有一定优势:1、间接法引入二抗,非特异反应多,背景高,二双抗原夹心法包被和标记都是特异性抗原,特异性强;2、间接法加样量一般20μL或以下,从而导致灵敏度低,而双抗原夹心法加样量高,一般50μL-100μL,一般无需稀释,灵敏度高;3、双抗原夹心法检测的是总抗,可以同时检测IgM抗体和IgG抗体,缩短窗口期。间接法一般检测IgG,窗口期较长;
双抗原夹心法检测可以分为一步法和两步法,一步法即为包被抗原、血清中的抗体和标记抗原混合反应后检测;两步法则是包被抗原先与血清中抗体反应,除杂后,包被抗原-血清中的抗体复合物再与标记抗原反应,最后检测。一步法反应步骤少,所用时间短,且包被抗原和标记抗原同时与血清中的抗体反应,出现抗体只与包被抗原或者标记抗原反应、夹心不成功的可能性小。但一步法会有钩状效应(HOOK效应)即当血清中抗体含量高时,会出现包被抗原和标记抗原量少于血清中抗体,导致抗原抗体不能成为夹心状态,导致强阳漏检。两步法反应步骤多,反应时间长,由于包被抗原和标记抗原分两步与血清中抗体反应,可能会出现包被抗原将抗体位点占据,无法形成夹心,低值灵敏度较一步法低,且背景较一步法高。但两步法由于有中间清洗步骤,将过多的抗体洗去,当血清中抗体较多时标记抗原仍能够与包被抗原-血清中抗体复合物连接形成夹心,避免钩状效应的产生。
因此,若能对双抗原夹心法进行技术改良,避免一步法和两步法的缺点,将有利于双抗原夹心法的进一步应用与推广。
发明内容
本发明的目的在于克服现有技术的缺陷,提供一种改良的双抗原夹心法模式,以综合一步法和两步法的优势。
具体的,为了实现本发明的上述目的,特采用以下技术方案:
本发明涉及一种双抗原夹心检测抗体的方法,所述方法以形成固相支持物-第一抗原Ag1-待检测抗体-第二抗原Ag2的形式完成检测,其中Ag2偶联有用于显示信号强度的标记物,所述方法包括以下步骤:
1)将Ag1、Ag2与待检物在足以发生抗体/抗原结合反应的条件下接触,形成免疫复合物;
其中以摩尔数计,Ag1的含量多于Ag2;
2)洗去未结合的待检测抗体;
3)加入Ag2并使其与所述免疫复合物中剩余的抗原结合位点结合;
4)检测所述标记物,以指示所述待检测抗体的存在和/或含量。
本发明进行两步反应,第一步反应时,同时加入包被抗原和适量标记抗原与血清中抗体反应,初步形成夹心,清洗后,再次加入标记抗原,与没有形成夹心但是已经形成包被抗原-血清中抗体复合物反应进一步形成夹心。
与现有技术相比,本发明采用双抗原夹心法检测,检测过程中采用两步法检测,且在第一步反应过程中加入适量酶标抗原,进而极大提高了检测的灵敏度,这样最终的试剂既保持了化学发光免疫分析灵敏度高、大批量快速自动检测、准确、重复性好、效期长并安全无毒无污染等优点,又能减少应用中内源性生物的干扰,同时降低研发成本,更易推广应用。
具体实施方式
本发明可通过后续对于本发明一些实施方案描述以及其中所包括的实施例的详细内容而更容易被了解。
在进一步叙述本发明之前,应明了本发明不会被局限于所述特定实施方案中,因为这些实施方案必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述特定实施方案,而非作为限制,因为本发明的范围将会被仅仅界定在所附的权利要求中。
本发明涉及一种双抗原夹心检测抗体的方法,所述方法以形成固相支持物-第一抗原Ag1-待检测抗体-第二抗原Ag2的形式完成检测,其中Ag2偶联有用于显示信号强度的标记物,所述方法包括以下步骤:
1)将Ag1、Ag2与待检物在足以发生抗体/抗原结合反应的条件下接触,形成免疫复合物;
其中以摩尔数计,Ag1的含量多于Ag2;
2)洗去未结合的待检测抗体;
3)加入Ag2并使其与所述免疫复合物中剩余的抗原结合位点结合;
4)检测所述标记物,以指示所述待检测抗体的存在和/或含量。
在一些实施方式中,所述方法用于非治疗用途。
本发明中使用的“抗原”指可在免疫反应中与抗体结合的任何物质。本发明中使用的抗原可为任何本领域已知的可用于免疫检测的抗原。
在一个实施方案中,抗原部分为与传染性疾病、内分泌、肿瘤或药物相关的抗原。在另一个实施方案中,抗原部分为病毒性或细菌性传染性疾病的相关抗原。在再一个实施方案中,抗原部分包括但不限于艾滋病毒抗原、甲型肝炎病毒抗原、乙型肝炎病毒抗原、丙型肝炎病毒抗原、丁型肝炎病毒抗原、戊型肝炎病毒抗原、庚型肝炎病毒抗原、风疹病毒抗原、人巨细胞病毒抗原、单纯疱疹病毒1型抗原、单纯疱疹病毒2型抗原、狂犬病毒抗原、人类T细胞白血病病毒抗原、登革热病毒抗原、人乳头瘤病毒抗原、西尼罗河病毒抗原、森林脑炎病毒抗原、麻疹病毒抗原、流感病毒抗原、副流感病毒抗原、水痘病毒抗原、艾柯病毒抗原、柯萨奇病毒抗原、乙型脑炎病毒抗原、柯萨奇病毒抗原、EB病毒抗原、腮腺炎病毒抗原、梅毒螺旋体抗原、包柔氏螺旋体抗原、沙眼衣原体抗原、肺炎衣原体抗原、鹦鹉热衣原体抗原、解脲脲原体抗原、肺炎支原体抗原、结核分枝杆菌抗原、幽门螺旋杆菌抗原、淋球菌抗原、疟原虫抗原、枯氏锥虫抗原、弓形虫抗原。
在一些实施方式中,在步骤1)中,以摩尔比计,Ag1:Ag2=6:2~6:5;也可以选择3:2或者2:1。
合适的Ag1与Ag2的添加比例能在保证灵敏度的前提下避免勾状效应。根据具体的抗原抗体特性不同,该比值可能有一些差别,但经过发明人验证,绝大多数抗原均满足上述比例。
在本发明中,“摩尔比”也可以为抗原的活性比,具有与待检测抗体结合的抗原表位的一个蛋白质可以认为是一个活性单位。
在一些实施方式中,以摩尔比计,步骤1)中加入的Ag2与步骤3)中加入的Ag2的比例为6:2~6:4。
在一些实施方式中,以摩尔比计,步骤1)中加入的Ag2与步骤3)中加入的Ag2的比例为2:1。
步骤3)中的Ag2以适当比例添加能够有效减少标记物的残留,以降低背景。
在一些实施方式中,所述固相支持物通过功能基团与Ag1非活性或低活性表位偶联;
和/或;
所述标记物通过功能基团与Ag2非活性或低活性表位偶联。
当抗原与磁珠和酶化学交联以后,构象容易发生变化,表位不易暴露,从而活性降低;故而在本发明的另一方面中,可通过优化的标记物偶联方式避免标记物对抗原活性的影响。
非活性表位是指偶联后不引起抗原构象变化的表位;
低活性表位是指偶联后在可接受程度内引起抗原构象变化的表位。
例如HCV病毒的抗原表位区段core的主要活性区富含赖氨酸,从而导致氨基残基在core活性区段富集,包被标记一般偶联的就是抗原的氨基,这样导致core活性表位失活或者被覆盖,从而活性降低;在这个例子中,氨基富集的表位多为活性表位;因而非氨基表位(例如羟基所在表位)可以认为是非活性/低活性表位(需验证)。
在一些实施方式中,所述功能基团在与Ag1/Ag2端连接处的官能团靶点选自羟基、羧基或氨基;
和/或;
所述功能基团在与所述固相支持物/所述标记物端连接处的官能团靶点为非选择性靶点,或选自羟基、羧基、氨基、巯基、醛基、DNA、RNA中的任一种。
功能基团可完全修饰在所述固相支持物/所述标记物上,功能基团的游离端与抗原表位的羟基、羧基或氨基偶联;
亦可以将功能基团在所述固相支持物/所述标记物上修饰一部分,在抗原表位处修饰一部分,再将二者偶联。
在一些实施方式中,所述功能基团选自下列反应基团:
芳基叠氮化物、碳化二亚胺、重氮甲烷、酰肼、羟甲基膦、亚胺酸酯、异氰酸酯、马来酰亚胺、盐乙酸酯、N-羟基琥珀酰亚胺酯、五氟代苯酯、补骨脂素、吡啶基二硫化物、乙烯基砜。
上述反应基团所对应的官能团靶点如下表所示:
在一些实施方式中,所述固相支持物为磁珠。
在一些实施方式中,所述磁珠为γFe2O3或Fe3O4磁性纳米粒子,或它们与有机高分子材料的复合体。
在一些实施方式中,所述标记物选自荧光物质、量子点、地高辛标记探针、生物素、放射性同位素、放射性造影剂、顺磁离子荧光微球、电子致密物质、化学发光标记物、超声造影剂、光敏剂、胶体金或酶中的任一种。
在一些实施方式中,所述荧光物质包括Alexa 350、Alexa 405、Alexa 430、Alexa488、Alexa 555、Alexa 647、AMCA、氨基吖啶、BODIPY 630/650、BODIPY 650/665、BODIPY-FL、BODIPY-R6G、BODIPY-TMR、BODIPY-TRX、5-羧基-4′,5′-二氯-2′,7′-二甲氧基荧光素、5-羧基-2′,4′,5′,7′-四氯荧光素、5-羧基荧光素、5-羧基罗丹明、6-羧基罗丹明、6-羧基四甲基罗丹明、Cascade Blue、Cy2、Cy3、Cy5、Cy7、6-FAM、丹磺酰氯、荧光素、HEX、6-JOE、NBD(7-硝基苯并-2-氧杂-1,3-二唑)、Oregon Green 488、Oregon Green 500、Oregon Green514、Pacific Blue、邻苯二甲酸、对苯二甲酸、间苯二甲酸、甲酚固紫、甲酚蓝紫、亮甲酚蓝、对氨基苯甲酸、赤藓红、酞菁、偶氮甲碱、花青、黄嘌呤、琥珀酰荧光素、稀土金属穴状化合物、三双吡啶基二胺铕、铕穴状化合物或螯合物、二胺、双花青苷、La Jolla蓝染料、别藻蓝蛋白、allococyanin B、藻蓝蛋白C、藻蓝蛋白R、硫胺、藻红青蛋白、藻红蛋白R、REG、罗丹明绿、罗丹明异硫氰酸酯、罗丹明红、ROX、TAMRA、TET、TRIT(四甲基罗丹明异硫醇)、四甲基罗丹明和德克萨斯红中的任一种。
在一些实施方式中,所述放射性同位素包括110In、111In、177Lu、18F、52Fe、62Cu、64Cu、67Cu、67Ga、68Ga、86Y、90Y、89Zr、94mTc、94Tc、99mTc、120I、123I、124I、125I、131I、154-158Gd、32P、11C、13N、15O、186Re、188Re、51Mn、52mMn、55Co、72As、75Br、76Br、82mRb和83Sr中的任一种。
在一些实施方式中,所述荧光微球为:聚苯乙烯荧光微球,内部包裹有稀土荧光离子铕。
在一些实施方式中,所述标记物选自辣根过氧化酶、碱性磷酸酶和葡萄糖氧化酶中的任一种。
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1
1).比较一步双抗原夹心法、两步双抗原夹心法和双抗原夹心综合法(实施例中内容)检测灵敏度。
为了对比三种反应模式的优劣,选取20例阴性样本和20例阳性标本同时采用两种反应模式进行检测,检测结果如下:
由下表可以看出,一步两步结合法检测灵敏度与一步法相当,较两步法更高,因此低值阳性测值更高,两步法对弱阳样本存在漏检风险;强阳样本测值一步法偏低,两步法和综合法测值较高,这是由于钩状效应导致,说明两步法和综合法可以避免钩状效应产生。因此双抗原综合法检测性能要优于一步法和双抗夹心直接两步法。
两种反应模式的比较结果
2).检出率比较
对比例的设置,将重组抗原替换为core+NS3抗原,其余同实施例。
测试200例确认为阳性的标本,验证试剂符合率,其中有2例有NS4表位抗原试剂检出,但不含NS4抗原漏检。
检测结果如下:
上表中,A为core+NS3抗原--试剂盒(对比例),B为core+NS3+NS4A+NS4B抗原--试剂盒(实施例)。
所述core、NS3、NS4A、NS4B的蛋白区段序列依次如SEQ ID NO:1-4所示。
从上表可知,丙型肝炎病毒(HCV)虽然在感染者体内滴度较低,但其几乎所有结构和非结构蛋白(NS2除外)都能够诱发机体的免疫应答,并产生相应的抗体。因而增加表位可以有效提高灵敏度,降低漏检风险。
3).活性表位比较
实验方案:
选取嵌合串联表达HCV抗原包被磁珠,应用不同交联方案,选取抗原不同残基交联,选取残基有:氨基、巯基、羟基。
AP标记抗人IgG抗体。
以单一变量实验原则,为了避免实验结果判断误差,酶免实验原理采用间接法,即磁珠包被抗原,AP标记二抗抗体。磁珠上的抗原连接血清中的抗HCV抗体,而后AP标记的抗抗体识别并捕获抗HCV抗体,形成磁珠-HCV抗原-标本中抗HCV抗体-抗抗体抗人IgG-AP复合物。清洗杂质后,复合物中的AP酶促AMPPD底物发光,发光值与标本中抗HCV抗体含量成正比。
具体酶免检测实验步骤:
1)取待测样本10uL加入包被有HCV重组嵌合抗原1的磁珠工作液50μL,同时加入测试处理液50uL,37℃孵育15min;
2)加入磁-场进行磁分离1min,去上清;
3)洗涤4次,每次250uL洗液,重复步骤2操作;
4)加入测试稀释液100μL,偶联了抗人IgG的碱性磷酸酶的工作液50uL,37℃孵育15min;
5)加入磁场进行磁分离1min,去上清;
6)洗涤4次,每次250uL洗液,重复步骤4操作;
7)加入底物100uL,必须于加化学发光底物液后的第5~30分钟内测量各孔的发光强度(RLU),测量时间1秒/孔。
8)均值各孔的RLU值与CUTOFF比较,样品测定值≥10×阴性质控,则判断为阳性,否则为阴性。
实验结果:
一般交联工艺选择蛋白巯基、氨基作为交联官能团,选择羧基官能团的交联工艺一般是采用EDC一步法,这套工艺同时连接了抗原的氨基和羧基,同时还有许多副反应,不好实现,一般不用。羟基官能团交联一般用在糖类等其他交联工艺上。
从上表中可以看出,羟基效果最好好。选取巯基时,NS3抗原失活风险较大,选取氨基时,core抗原失活风险较大。
实施例2
本实施例涉及一种梅毒螺旋体的检测方法。
1.试剂盒的制备。
本发明的梅毒螺旋体抗体化学发光免疫分析测定试剂盒:包括以下步骤:
1)梅毒螺旋体特异性重组嵌合抗原的制备:
所述梅毒螺旋体特异性重组嵌合抗原由TpN15、TpN17、TpN47和TmpA中的两种或多种抗原通过分子生物学方法连接而来,更优选为TpN15、TpN17和TpN47中的两种或三种,上述嵌合抗原转化到大肠杆菌中使其表达,最后通过蛋白纯化技术得到梅毒螺旋体特异性嵌合蛋白抗原。
2)磁珠体系:
磁珠为Fe2O3或Fe3O4磁性纳米粒子与有机高分子材料的复合体,并具有0.1-5μm的粒径,其表面带有-COOH功能基团。根据活性功能团可通过EDC等化学试剂将梅毒螺旋体特异性重组嵌合抗原连接于磁珠上。进一步的,可采用以下方法制备:
a)将梅毒螺旋体特异性重组嵌合抗原用1×PBS进行透析;
b)将透析后梅毒螺旋体特异性重组嵌合抗原,以5-100ug/mg磁珠的比例和活化后的磁珠进行混合,优选20-50ug/mg磁珠的用量,然后加入EDC(MES5.0缓冲液溶解),优选0.05-0.2mg/mg磁珠,2~8℃反应18小时;
c)将反应物用磁分离器分离,用磁珠试剂缓冲液将磁珠稀释后作为工作液使用。
3)酶标记物体系:
在其中一个实施例中,所述酶示踪物选自碱性磷酸酶,进一步可采用以下方法制备:
a)将检测嵌合抗原用0.1倍PBS进行透析,然后用2-IT进行活化;
b)将碱性磷酸酶用SMCC进行活化;
c)将活化后的检测抗原和活化后的碱性磷酸酶进行混合,2~8℃反应6-24小时。
d)将反应物用层析柱纯化,用酶标试剂缓冲液将酶标抗原稀释后作为工作液使用。所述梅毒螺旋体重组嵌合抗原和碱性磷酸酶的质量比为1:1-1:20,2-IT与抗原的摩尔比为1:1-1:100,SMCC与碱性磷酸酶的摩尔比为1:1-1:100。
4)发光底物制备
人工合成金刚烷胺类发光底物及其增强剂,均经无菌过滤,制备发光底物工作液。
5)洗涤液制备
洗涤液为含有Tween-20的PBS,其中Tween-20的工作浓度为0.01-0.1%,优选0.03-0.05%。
6)质控品
梅毒螺旋体特异性抗体阴性质控品:由非梅毒感染的健康人群血清配制而成。
梅毒螺旋体特异性抗体阳性质控品:由梅毒患者的特异性抗体阳性血清配制而成;
7)制备梅毒螺旋体抗体高通量检测试剂盒
包被梅毒螺旋体特异性重组嵌合抗原的磁珠、碱性磷酸酶标记梅毒螺旋体特异性重组嵌合抗原、发光底物、洗涤液、梅毒螺旋体特异性抗体阴性质控品、梅毒螺旋体特异性抗体阳性质控品和外包装盒共同组成的梅毒螺旋体特异性抗体化学发光检测试剂盒。
2.梅毒螺旋体抗体的检测方法,采用上述的试剂盒,包括以下步骤:
1)取待测样本50uL加入包被有梅毒螺旋体特异性重组嵌合抗原的磁性微球25uL,同时加入酶标抗原100ul,37℃孵育15min;
2)加入磁-场进行磁分离1min,去上清;
3)洗涤4次,每次250uL洗液,重复步骤2操作;
4)加入酶标试剂100uL,孵育15min;
5)加入磁场进行磁分离1min,去上清;
6)洗涤4次,每次250uL洗液,重复步骤4操作;
7)加入底物100uL,必须于加化学发光底物液后的第5~30分钟内测量各孔的发光强度(RLU),测量时间1秒/孔。
8)均值各孔的RLU值与CUTOFF比较,样品测定值≥20×阴性质控,则判断为阳性,否则为阴性。
为了对比两种反应模式的优劣,选取20例阴性样本和20例阳性标本同时采用两种反应模式进行检测,检测结果如下:
两种反应模式的比较结果
由表一可以看出,一步两步结合法检测灵敏度更高,因此阳性测值更高,直接两步夹心法对部分弱阳标本存在漏检情况,而一步两步结合法对这部分弱阳标本反应信号有明显增强。因此一步两步结合法检测性能要优于直接两步双抗夹心法。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
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Claims (10)
1.一种双抗原夹心检测抗体的方法,所述方法以形成固相支持物-第一抗原Ag1-待检测抗体-第二抗原Ag2的形式完成检测,其中Ag2偶联有用于显示信号强度的标记物,其特征在于,包括以下步骤:
1)将Ag1、Ag2与待检物在足以发生抗体/抗原结合反应的条件下接触,形成免疫复合物;
其中以摩尔数计,Ag1的含量多于Ag2;
2)洗去未结合的待检测抗体;
3)加入Ag2并使其与所述免疫复合物中剩余的抗原结合位点结合;
4)检测所述标记物,以指示所述待检测抗体的存在和/或含量。
2.根据权利要求1所述的双抗原夹心检测抗体的方法,其特征在于,在步骤1)中,以摩尔比计,Ag1:Ag2=6:2~6:5。
3.根据权利要求2所述的双抗原夹心检测抗体的方法,其特征在于,以摩尔比计,步骤1)中加入的Ag2与步骤3)中加入的Ag2的比例为6:2~6:4。
4.根据权利要求1所述的双抗原夹心检测抗体的方法,其特征在于,所述固相支持物通过功能基团与Ag1非活性或低活性表位偶联;
和/或;
所述标记物通过功能基团与Ag2非活性或低活性表位偶联。
5.根据权利要求4所述的双抗原夹心检测抗体的方法,其特征在于,所述功能基团在与Ag1/Ag2端连接处的官能团靶点选自羟基、羧基或氨基;
和/或;
所述功能基团在与所述固相支持物/所述标记物端连接处的官能团靶点为非选择性靶点,或选自羟基、羧基、氨基、巯基、醛基、DNA、RNA中的任一种。
6.根据权利要求5所述的双抗原夹心检测抗体的方法,其特征在于,所述功能基团选自下列反应基团:
芳基叠氮化物、碳化二亚胺、重氮甲烷、酰肼、羟甲基膦、亚胺酸酯、异氰酸酯、马来酰亚胺、盐乙酸酯、N-羟基琥珀酰亚胺酯、五氟代苯酯、补骨脂素、吡啶基二硫化物、乙烯基砜。
7.根据权利要求4~6任一项所述的双抗原夹心检测抗体的方法,其特征在于,所述固相支持物为磁珠。
8.根据权利要求7所述的双抗原夹心检测抗体的方法,其特征在于,所述磁珠为γFe2O3或Fe3O4磁性纳米粒子,或它们与有机高分子材料的复合体。
9.根据权利要求4~6任一项所述的双抗原夹心检测抗体的方法,其特征在于,所述标记物选自荧光物质、量子点、地高辛标记探针、生物素、放射性同位素、放射性造影剂、顺磁离子荧光微球、电子致密物质、化学发光标记物、超声造影剂、光敏剂、胶体金或酶中的任一种。
10.根据权利要求9所述的双抗原夹心检测抗体的方法,其特征在于,所述标记物选自辣根过氧化酶、碱性磷酸酶和葡萄糖氧化酶中的任一种。
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