CN117147819A - 浓度估计试剂盒和浓度估计方法 - Google Patents
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Abstract
一种浓度估计试剂盒,包括衍生化试剂,所述衍生化试剂衍生混合试样中包括的抗原的至少一部分,并且所述衍生化试剂用于获得具有不同衍生化率的多个样本,所述衍生化率是衍生的抗原相对于试样中包括的抗原的比率;与抗原结合的抗体;和用染料修饰的抗原。
Description
相关申请的交叉引用
本申请要求2022年6月1日提交的日本专利申请号2022-89389的权益,其全部公开内容通过引用的方式并入本文。
技术领域
本公开总体上涉及浓度估计试剂盒和浓度估计方法。
背景技术
荧光偏振免疫分析(FPIA)法是一种使用荧光的免疫分析方法。在FPIA中测量的荧光偏振度与待测物质的有效体积成比例。日本未审查的专利申请公开号H03-103765描述了一种FPIA,其使用由于试剂和荧光标记的抗原之间的特异性抗原-抗体反应而引起的荧光偏振度改变的特征,在所述试剂中,抗体固定在具有比抗体更大分子量的物质上。
FPIA使用与待测物质抗原特异性结合的抗体。为了获得这种抗体,待测物质必须具有免疫原性,即诱导抗体产生的活性。当与抗体结合但由于其分子量低而本身无免疫原性的半抗原是待测物质时,必须使半抗原具有免疫原性才能获得抗体。例如,日本未审查的专利申请公开号S51-104029公开了,为了定量安替比林,使用通过将安替比林与血清白蛋白结合而获得的安替比林衍生物作为抗原制备的抗体,进行基于抗原-抗体反应的测定。
在FPIA中,检测与抗原和抗体的结合相对应的荧光偏振度的变化,但是与抗体结合的抗原即待测物质的浓度范围是有限的。因此,在FPIA中,根据基于荧光偏振度的测量值的校准曲线可估计的待测物质的浓度范围取决于待测物质和抗体之间的亲和力。当没有与待测物质的亲和力不同的多种抗体时,待测物质的浓度的可估计范围受到限制。
鉴于上述情况做出本公开,并且本公开的目的是提供浓度估计试剂盒和浓度估计方法,藉此可以扩大待测量物质的浓度的可估计范围。
发明内容
根据本公开的第一方面的浓度估计试剂盒包括:
衍生化试剂,所述衍生化试剂衍生混合试样中包括的抗原的至少一部分,并且所述衍生化试剂用于获得具有不同衍生化率的多个样品,所述衍生化率是衍生的抗原相对于试样中包括的抗原的比率;
与抗原结合的抗体;和
用染料修饰的抗原。
根据本公开的第二方面的浓度估计方法包括:
混合包括抗原的试样和衍生所述试样中包括的至少一部分抗原的衍生化试剂,并获得具有不同衍生化率的多个样品,所述衍生化率是衍生的抗原相对于试样中包括的抗原的比率;
将各样品、与抗原结合的抗体和用染料修饰的抗原混合以获得多个待测溶液;和
测量各待测溶液的偏振度。
应当理解,前面的概述和下面的详细描述都是示例性和解释性的,并不限制本公开。
根据本公开,可以扩大待测物质的浓度的可估计范围。
附图说明
当结合以下附图考虑以下具体实施方式时,可以获得对本申请更完整的理解,其中:
图1是说明当改变抗体的浓度和衍生化率时,与抗体结合的抗原相对于抗原的比率的图;
图2是说明从衍生化率计算的偏振度的图;
图3是示意性地说明包括示踪剂和衍生化试剂的多孔板的图,所述示踪剂是用染料修饰的抗原,所述衍生化试剂具有按行不同的物质量;
图4是说明根据实施例的荧光偏振度测量设备的配置的图;
图5是说明有效视野中的微通道的图;和
图6是说明相对于具有不同酰化试剂添加量的样品的组胺浓度的偏振度的图。
具体实施方式
参考附图描述了本公开的实施方案。注意,本公开不限于下面描述的实施例和附图。此外,注意,在以下实施方案中,表达“具有”和“包括”或“包含”还包括“含有”或“由...组成”的含义
根据本实施方案的浓度估计试剂盒是用于FPIA的试剂盒,其中使用抗体与特定抗原的结合来估计试样中作为待测物质的抗原的浓度。为便于描述,以下将试样中的抗原称为“待测物质”。对试样没有特别限制,只要试样是待检验或分析的物质。试样的实例包括细胞、组织、细胞培养上清液、细胞提取物、组织提取物、体液如从人或非人动物获得的血液、唾液、尿和淋巴、生物样品如鼻或鼻咽拭子、饮料、食物、物体清洁液等。
根据本实施方案的浓度估计试剂盒包括衍生化试剂、与待测物质结合的抗体和用染料修饰的抗原。衍生化试剂对混合样品中包含的至少一部分待测物质进行衍生。这里,术语“衍生化”是指除了构成待测物质的氢原子之外,使用诸如羟基、氨基、羧基、巯基、羰基和巯基的官能团来向待测物质添加取代基。衍生化的实例包括甲硅烷化、酰化、酯化、肟化等。衍生化可以采用使用已知的交联剂向待测物质中添加取代基的形式。所述取代基是任何可以用已知方法取代构成待测物质的原子的取代基。取代基的例子包括酰基、烷基等,其中酰基是优选的。当要向待测物质中加入酰基时,衍生化试剂的实例包括用酰基(RCO-)取代待测物质的氢原子如羟基、氨基和巯基的酰化试剂(酰化剂)。酰化试剂的实例包括酰氯、酸酐、乙烯酮、羧酸等,酰化试剂的具体实施例包括三氟乙酸酐、三氟乙酸咪唑、4-氯丁酰氯等。甲硅烷化试剂的实例包括六甲基二硅氮烷(HMDS)、N-三甲基甲硅烷基咪唑等。酯化试剂的实例包括酸-醇、N,N-二甲基甲酰胺、二甲基缩醛、柱上甲基化剂、重氮甲烷等。肟化试剂的实例包括五氟苄基、羟胺盐酸盐等。交联剂的实例包括醛、酮等。在一个实例中,交联剂是戊二醛。
衍生化试剂用于获得多个具有不同衍生化率的样品。这里,衍生化率是待测衍生的物质相对于试样中所含待测物质的比率。衍生化率取决于衍生化试剂的量(物质量)相对于试样中包含的待测物质的量(物质量)。假设衍生化试剂是以具有不同物质量的衍生化试剂的多个溶液的形式存在,当估计相同试样中待测物质的浓度时,通过混合具有相同体积的各试样和具有不同物质量的衍生化试剂的多个溶液,可以获得具有不同衍生化率的多个样品。例如,具有不同物质量的衍生化试剂的多个溶液可以包括物质量为最小值M的溶液,以及衍生化试剂的物质量为M的正实倍数的且物质量不同的多个溶液。衍生化试剂可以是包括相同浓度的衍生化试剂且具有不同体积的多个溶液,或者可以是包括不同浓度的衍生化试剂且具有相同体积的多个溶液。
浓度估计试剂盒可包括一种非衍生化试剂,用于获得衍生化率为0的样品。当浓度估计试剂盒包括含有相同浓度和不同体积的衍生化试剂的多个溶液时,浓度估计试剂盒可以包括含有与含有衍生化试剂的溶液相同的溶剂并且不含有衍生化试剂的非衍生化试剂。当浓度估计试剂盒包括含有不同浓度和相同体积的衍生化试剂的多个溶液时,浓度估计试剂盒可以包括含有与含有衍生化试剂的溶液相同体积的相同溶剂,并且不含有衍生化试剂的非衍生化试剂。
只要抗体与待测物质特异性结合,抗体不受限制。抗体的实例包括单克隆抗体、多特异性抗体、双功能抗体、人抗体、人源化抗体、来自鸟类如鸡、非灵长类如骆驼、非人类哺乳动物和其它动物的抗体、重组抗体、嵌合抗体、单链Fv、单链抗体、单结构域抗体、Fab片段、F(ab′)片段、F(ab′)2片段、二硫键连接的Fv、抗独特型抗体、双结构域抗体、双可变结构域抗体等。
只要能够通过用衍生化试剂进行衍生化来改变与抗体的亲和性,对待测物质没有特别限制。考虑到衍生化的效率,优选待测物质是低分子量物质,而不是高分子量物质如蛋白质等。优选待测物质为半抗原。半抗原是一种与抗体结合的物质,但由于其分子量低,其本身不表现出免疫原性,即诱导抗体产生的活性。半抗原的实例包括组胺、γ-氨基丁酸(GABA)、多巴胺、甲状腺激素、类固醇激素等。更具体地,甲状腺激素是三碘甲状腺原氨酸、甲状腺素、3,5-二碘-L-甲状腺原氨酸等。类固醇激素是雌酮、雌二醇、雌三醇、孕酮、皮质醇、睾酮、硫酸脱氢表雄酮等。半抗原可以是低分子量肽激素、儿茶酚胺、外稃酶维生素、药物、抗生素、其代谢物等。
半抗原通过与蛋白质等免疫原性物质结合而成为免疫原性完全的抗原。免疫原性物质的实例包括免疫原性蛋白质、多肽、碳水化合物、多糖、脂多糖、核酸等。免疫原性物质优选为蛋白质或多肽,其实例包括牛血清白蛋白(BSA)、匙孔血蓝蛋白(KLH)和甲状腺球蛋白。
当待测物质是半抗原时,优选抗体是其免疫原是半抗原衍生物的抗体,其中免疫原性物质通过接头与所述半抗原结合。接头是在免疫原性物质和半抗原之间引入的基团。接头的实例包括包含酰胺、二硫化物、硫醚、腙、酰肼、亚胺、肟、脲、硫脲、脒、胺、磺酰胺等的接头。
其免疫原是半抗原衍生物((半抗原)-(接头)-(免疫原性物质))的抗体可以通过已知方法获得。获得抗体时,通常,向宿主动物如兔、山羊、小鼠、豚鼠、马等注射免疫原即可。为了增强免疫原性,优选注射免疫原和佐剂的混合物。可以在宿主动物的同一部位或不同部位定期或不定期地进一步注射免疫原。适当评价抗体滴度,通过从宿主动物等采集血液,能够回收与半抗原衍生物特异性结合的抗体。
当待测物质是半抗原并且使用其免疫原是半抗原衍生物的抗体时,优选衍生化试剂赋予分析物中的半抗原与接头的至少一部分相同的结构。例如,当接头含有酰基时,使用赋予半抗原酰基的衍生化试剂即可。
根据本实施方案的浓度估计试剂盒的抗原用染料修饰,并且在免疫测定中用作示踪剂。在下文中,浓度估计试剂盒的用染料修饰的抗原也称为“示踪剂”。优选发射荧光的荧光染料作为染料。每种荧光染料都有荧光寿命。根据待测物质的分子量等适当选择荧光寿命为1至10ns的荧光染料、荧光寿命大于10ns至200ns的荧光染料或荧光寿命大于200ns至3000ns的荧光染料即可。荧光寿命为1至10ns的荧光染料的实例包括荧光素化合物,例如吲哚宁、氯三嗪基氨基荧光素、4’-氨基甲基荧光素、5-氨基甲基荧光素、6-氨基甲基荧光素、6-羧基荧光素、5-氨基荧光素、6-氨基荧光素、硫脲荧光素、甲氧基三嗪基荧光素等;罗丹明B、罗丹明6G、罗丹明6GP等罗丹明衍生物;以及,作为注册商标或商品名称的Alexa Fluor系列,例如Alexa Fluor 488、BODIPY系列、DY系列、ATTO系列、Dy Light系列、Oyster系列、HiLyte Fluor系列、Pacific Blue、Marina Blue、吖啶、Edans、香豆素、DANSYL、FAN、OregonGreen、罗丹明Green-X、NBD-X、TET、JOE、Yakima Yellow、VIC、HEX、R6G、Cy3、TAMRA、罗丹明Red-X、Redmond Red、ROX、Cal Red、Texas Red、LC Red 640、Cy5、Cy5.5和LC Red705。荧光寿命大于10ns至200ns的荧光染料的实例包括萘衍生物如二烷基氨基萘磺酰基等,和芘衍生物如N-(1-芘基)马来酰亚胺、氨基芘、芘丁酸、炔基芘等。荧光寿命大于200ns至3000ns的荧光染料的实例包括金属络合物,例如铂、铼、钌、锇、铕等。
为了用染料修饰抗原,例如,染料和抗原直接共价键合或通过接头如低聚乙二醇、烷基链等键合即可。当抗原是半抗原,并且使用其免疫原是半抗原衍生物的抗体时,优选染料通过与插入半抗原和半抗原衍生物中的免疫原性物质之间的接头的至少一部分相同的结构与半抗原结合。例如,当插入半抗原和半抗原衍生物中的免疫原性物质之间的接头含有酰基时,优选插入染料和抗原之间的接头含有酰基。
所述染料具有能够与抗原的羧基、氨基、羟基、巯基、苯基等结合的官能团。通过使染料和抗原的相应官能团在已知条件下反应,可以用染料标记抗原。注意,可以根据需要选择修饰抗原的一个分子的染料分子的数目。优选每个抗原分子有一个或多个染料分子,并且可以是2至5个分子。
接下来,描述使用上述浓度估计试剂盒的根据本实施方案的浓度估计方法的实例。浓度估计方法包括样品制备步骤、混合步骤和测量步骤。在样品制备步骤中,将含有上述待测物质的试样和衍生化试剂混合,获得具有不同衍生化率的多个样品。所述多个样品可以通过将试样与含有相同浓度和不同体积的衍生化试剂的多个溶液中的每一种混合而获得,或者可以通过将试样与含有不同浓度和相同体积的衍生化试剂的多个溶液中的每一种混合而获得。例如,将相同量的试样分配到多孔板的各个孔中,并将每种溶液添加到各个孔中即可。
在混合步骤中,将各样品、抗体和示踪剂混合,获得多个待测溶液。如果使用上述多孔板,则向存在各样品的各孔中加入抗体和示踪剂即可。
在测量步骤中,测量各个待测溶液的偏振度。FPIA使用基于示踪剂与抗体结合来形成示踪剂-抗体缀合物而引起的示踪剂分子量变化的偏振度变化。当在激发态保持稳态时,溶液中的染料在同一平面上发射偏振荧光,但是当在激发态以布朗运动旋转时,在不同于激发平面的平面上发射荧光,因此荧光被去偏振。荧光偏振度表示荧光分子在从激发到发射荧光的一段时间内旋转了多少。由于布朗运动,低分子量分子在溶液中剧烈旋转,因此偏振度较低。高分子量分子具有弱布朗运动,因此,偏振度升高。例如,在通过混合特异性结合待测物质A的抗体B和通过用荧光染料标记待测物质A获得的示踪剂C而获得的溶液中,待测物质A和示踪剂C在溶液中与抗体B竞争性反应。因此,当待测物质A的浓度高时,与抗体B结合的待测物质A的量增加(与抗体B结合的示踪剂C的量减少),与抗体B未结合的游离示踪剂C的量增加。同时,当待测物质A的浓度低时,与抗体B结合的待测物质A减少(与抗体B结合的示踪剂C增加),与抗体B未结合的游离示踪剂C减少。当游离示踪剂C的质量与示踪剂C与抗体B结合形成的缀合物的质量之间存在差异时,可以使用偏振度的变化作为指标来测量待测物质A的浓度。
在FPIA中,由示踪剂与待测物质结合引起的分子量变化被测量为分子取向的时间变化。可以使用期望的偏振测量设备来测量偏振度。在反应结束后的预定时间内测量偏振度即可。为了定量待测物质,创建校准曲线并与样品的测量值进行比较即可,所述校准曲线是通过使用含有已知浓度的待测物质的溶液执行与上述相同的操作而预先获得的。
利用根据本实施方案的浓度估计试剂盒和浓度估计方法,测量从具有不同衍生化率的多个样品制备的每个待测溶液的偏振度,以估计试样中的待测物质的浓度。当Ag_A为待测衍生化物质,Ag_B为待测未衍生化物质,Ab为抗体,Ag_A与抗体的偶联物为Ag_A-Ab,Ag_B与抗体的偶联物为Ag_B-Ab时,Ag_A与Ab的结合常数KA、Ag_B与Ab的结合常数KB表示如下:
方程1
当BF_A和BF_B分别是Ag_A的结合/游离比和Ag_B的结合/游离比时,得到以下结果。
方程2
当pA、pB和q分别是Ag_A的引入量(M)、Ag_B的引入量(M)和Ab的引入量(M)时,BF_A可以使用以下三次方程来计算。
方程3
基于所述三次方程,在改变衍生化率和抗体浓度的同时,估计结合到抗体的抗原相对于抗原(衍生化抗原和非衍生化抗原的总量)的比率,结果显示,如图1所示,当衍生化率改变时,抗原对抗体的亲和力改变(KA:2E+9M-1,KB:2E+6M-1和抗原总量(pA+pB):1E-8M)。
在FPIA中,测量示踪剂的偏振度。假设示踪剂与抗体的结合常数与衍生的抗原的结合常数KA相同。示踪剂的B/F比相当于衍生的抗原的B/F比(BF_A)。当Fh是示踪剂与抗体结合时的偏振度,Fl是示踪剂游离时的偏振度时,FPIA系统中示踪剂的偏振度P用P=(Fh×BF_A+Fl)/(1+BF_A)表示。图2示出了对于其中KA、KB、抗体浓度、示踪剂浓度、Fh和Fl分别是2E+9M-1、2E+6M-1、1E-7M、1E-8M、300mP和100mP的情况,根据衍生化率计算的偏振度。图2表明,当衍生化率不同时,偏振度变化的抗原浓度范围有变化。
根据本实施方案的浓度估计试剂盒,通过测定含有待测物质且衍生化率不同的多个样品的偏振度,能够改变待测物质对抗体的亲和性。在FPIA中,可测量浓度的范围取决于待测物质对抗体的亲和力,因此,可扩大待测物质的可估计浓度范围。
注意,其中浓度估计试剂盒包括多孔板的配置是可能的,其中衍生化试剂以不同的物质量固定在多孔板的各个孔中。衍生化试剂可以通过已知方法固定在孔中。例如,将含有衍生化试剂的溶液加入孔中,并通过干燥等除去溶剂即可。
图3示意性地示出了上述多孔板。多孔板包括3×3排列的孔。抗体、示踪剂和衍生化试剂固定在每个孔中。所有孔中固定化抗体和示踪剂的物质量相同。然而,固定的衍生化试剂的物质量按行不同,第一行、第二行和第三行的孔中固定的衍生化试剂的物质量分别为M1、M2和M3。
以柱A为例,通过向柱A的三个孔中各加入相同体积的试样,就可以根据衍生化试剂的物质量衍生试样中所含的待测物质。通过使用上述多孔板,可以容易地获得具有不同衍生化率的多个样品。多孔板包括柱B和柱C,柱B和柱C具有与柱A相似的衍生化试剂的不同的物质量,因此可用于不同试样的试验、一式两份的试验、一式三份的试验等。注意,板的孔的数量可以大于9个,并且根据孔的数量适当地设置行和列的数量。
实施例
在下文中,使用实施例来进一步详细描述本公开,但是本公开不限于这些实施例。
使用5/6-TAMRA(罗丹明)对组胺(由Fuji Film Wako Pure Chemical公司生产)进行修饰以获得组胺示踪剂。将组胺示踪剂溶于纯水中,得到2nM溶液。用磷酸盐缓冲液(PBS)稀释抗组胺抗体(由Progen Biotechnik生产)以制备21nM溶液。将组胺(由Fuji Film WakoPure Chemical生产)溶于纯水中以制备33mg/mL的溶液。向四个试管各自引入100μL获得的组胺溶液,并向各试管中加入RIDA筛选组胺ELISA试剂盒(R-biopham制造)中包含的酰化试剂。酰化试剂的添加量为25μL(样品1)、10μL(样品2)和5μL(样品3)。其中一个试管中不添加酰化试剂(样品4)。
用水将样品1至4稀释10倍的操作进行8次。因此,获得了样品1至4的样品的九个水平。将25μL的获得的9个水平的样品、25μL的组胺示踪剂和25μL的抗体溶液混合,并在遮光环境中于室温静置60分钟,然后测量每种混合溶液的荧光偏振度。
使用配有9个微通道的荧光偏振度测量装置测量荧光偏振度。图4中示出了所使用的荧光偏振度测量设备10的配置。荧光偏振度测量装置10包括LED光源1、激发滤光器2、物镜3、样品光发射器4、二向色滤光器5、荧光滤光器6、数字成像元件(CMOS或CCD)7、成像透镜8和液晶元件9。来自LED光源1的中心波长为565nm的激发光通过激发滤光器2和物镜3发射到样品光发射器4中的样品上,样品发射的荧光通过二向色滤光器5和荧光滤光器6透射,并且数字成像元件7获取透射光。透射荧光的偏振方向可以通过调制施加到设置在荧光滤光器6和图像形成透镜8之间的液晶元件9的电压来调制。通过使该调制频率与数字成像元件7的捕获频率同步来获取和计算图像,并且偏振度P被提取为二维图像。
荧光偏振度测量装置10的样品光发射器4的光学观察部分的有效视野的直径约为3mm。如图5所示,通道11和通道12之间的空间在圆所示的有效视野的3mm直径内以相等的间隔提供。通道的宽度为200μm,通道之间的间距为100μm。通道的深度为900μm。由于在样品光发射器4中形成多个微通道,因此可以同时测量多个样品。激发波长为546±11nm,检测波长为590±16.5nm。将9个水平的样品制备的混合溶液分别注入9个微通道,同时进行测定。
结果
图6示出了对于具有不同酰化试剂添加量的样品1至4,相对于抗原(组胺)浓度的偏振度。偏振度改变的抗原浓度范围根据酰化试剂的添加量而改变。研究表明,通过使用具有不同衍生化率的多个样品,可以扩大组胺浓度的可估计范围。
出于解释的目的,前面描述了一些示例实施方案。尽管前面的讨论已经给出了具体的实施方案,但是本领域技术人员将认识到,在不脱离本发明的更广泛的精神和范围的情况下,可以在形式和细节上进行改变。因此,说明书和附图被认为是说明性的,而不是限制性的。因此,该详细描述不应被认为是限制性的,并且本发明的范围仅由所包括的权利要求以及这些权利要求所赋予的等同物的全部范围来限定。
Claims (10)
1.一种浓度估计试剂盒,其包括:
衍生化试剂,所述衍生化试剂衍生混合试样中包括的抗原的至少一部分,并且所述衍生化试剂用于获得具有不同衍生化率的多个样本,所述衍生化率是衍生的抗原相对于试样中包括的抗原的比率;
与抗原结合的抗体;和
用染料修饰的抗原。
2.根据权利要求1所述的浓度估计试剂盒,其中
所述抗原是半抗原,
所述抗体是其免疫原是半抗原衍生物的抗体,所述半抗原衍生物中免疫原性物质通过接头与所述半抗原结合,
所述衍生化试剂赋予试样中的半抗原与接头的至少一部分相同的结构,和
所述染料通过所述与接头的至少一部分相同的结构与所述半抗原结合。
3.根据权利要求2所述的浓度估计试剂盒,其中
所述半抗原是组胺。
4.根据权利要求1至3中任一项所述的浓度估计试剂盒,其中
所述衍生化试剂是包括相同浓度的衍生化试剂且具有不同体积的多个溶液。
5.根据权利要求1至3中任一项所述的浓度估计试剂盒,其中
所述衍生化试剂是包括不同浓度的衍生化试剂且具有相同体积的多个溶液。
6.一种浓度估计方法,其包括:
混合包括抗原的试样与衍生所述试样中包括的至少一部分抗原的衍生化试剂,并获得具有不同衍生化率的多个样品,所述衍生化率是衍生的抗原相对于试样中包括的抗原的比率;
将各个样品、与抗原结合的抗体和用染料修饰的抗原混合以获得多个待测溶液;和
测量每个待测溶液的偏振度。
7.根据权利要求6所述的浓度估计方法,其中
所述抗原是半抗原,
所述抗体是其免疫原是半抗原衍生物的抗体,所述半抗原衍生物中免疫原性物质通过接头与所述半抗原结合,
所述衍生化试剂赋予试样中的半抗原与接头的至少一部分相同的结构,和
所述染料通过所述与接头的至少一部分相同的结构与所述半抗原结合。
8.根据权利要求7所述的浓度估计方法,其中
所述半抗原是组胺。
9.根据权利要求6至8中任一项所述的浓度估计方法,其中
所述样品的制备包括通过混合多个溶液获得多个样品,所述多个溶液包括相同浓度的衍生化试剂,并且具有不同的体积。
10.根据权利要求6至8中任一项所述的浓度估计方法,其中
所述样品的制备包括通过混合多个溶液获得多个样品,所述多个溶液包括不同浓度的衍生化试剂,并且具有相同的体积。
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