Siemens Healthcare Diagnostics, Advanced Technology and Pre-Development, 333 Coney Street, East Walpole, MA 02032, USA.
Org Biomol Chem. 2013 Feb 14;11(6):1026-39. doi: 10.1039/c2ob27190g. Epub 2013 Jan 7.
Chemiluminescent acridinium dimethylphenyl esters containing N-sulfopropyl groups in the acridinium ring are highly sensitive, hydrophilic labels that are used in automated immunoassays for clinical diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of a cationic surfactant. At physiological pH, N-sulfopropyl acridinium esters exist as water adducts that are commonly referred to as pseudobases. Pseudobase formation, which results from addition of water to the zwitterionic N-sulfopropyl acridinium ring, neutralizes the positive charge on the acridinium nitrogen and imparts a net negative charge to the label due to the sulfonate moiety. As a consequence, N-sulfopropyl acridinium ester conjugates of small molecule haptens as well as large molecules such as proteins gain negative charges at neutral pH. In the current study, we describe the synthesis and properties of two new hydrophilic acridinium dimethylphenyl ester labels where the net charge in the labels was altered. In one label, the structure of the hydrophilic N-alkyl group attached to the acridinium ring was changed so that the pseudobase of the label contains no net charge. In the second acridinium ester, two additional negative charges in the form of sulfopropyl groups were added to the acridinium ring to make this label's pseudobase strongly anionic. Chemiluminescence measurements of these labels, as well as their conjugates of an antibody with a neutral pI, indicate that acridinium ester charge while having a modest effect on emission kinetics has little influence on light output. However, our results demonstrate that acridinium ester charge can affect protein pI, apparent chemiluminescence stability and non-specific binding of protein conjugates to microparticles. These results emphasize the need for careful consideration of acridinium ester charge in order to optimize reagent stability and performance in immunoassays. In the current study, we observed that for a neutral protein, an acridinium ester with a hydrophilic but charge-neutral N-alkyl group afforded faster light emission, lower non-specific binding and better chemiluminescence stability than an analogous label with an anionic N-alkyl group.
含 N-磺丙基的化学发光吖啶二甲基苯基酯在吖啶环中是高度敏感的亲水性标记物，用于临床诊断的自动化免疫分析。在存在阳离子表面活性剂的情况下，碱性过氧化物会引发这些标记物的发光。在生理 pH 值下，N-磺丙基吖啶酯以水加合物的形式存在，通常称为假碱基。假碱基的形成是由于水与两性离子 N-磺丙基吖啶环加成，使吖啶氮上的正电荷中和，并由于磺酸盐部分使标记物带净负电荷。因此，小分子半抗原和蛋白质等大分子的 N-磺丙基吖啶酯缀合物在中性 pH 值下获得负电荷。在当前的研究中，我们描述了两种新的亲水性吖啶二甲基苯基酯标记物的合成和性质，其中改变了标记物的净电荷。在一种标记物中，连接到吖啶环上的亲水性 N-烷基的结构发生变化，使得标记物的假碱基没有净电荷。在第二种吖啶酯中，在吖啶环上添加了两个额外的负电荷磺酸丙基，使该标记物的假碱基带强烈的阴离子电荷。这些标记物及其与具有中性 pI 的抗体的缀合物的化学发光测量表明，吖啶酯电荷虽然对发射动力学有适度的影响，但对光输出影响不大。然而，我们的结果表明，吖啶酯电荷可以影响蛋白质的 pI、表观化学发光稳定性和蛋白质缀合物对微球的非特异性结合。这些结果强调了在免疫分析中为了优化试剂稳定性和性能，需要仔细考虑吖啶酯电荷。在当前的研究中，我们观察到对于中性蛋白质，具有亲水性但电荷中性的 N-烷基的吖啶酯比具有阴离子 N-烷基的类似标记物具有更快的发光、更低的非特异性结合和更好的化学发光稳定性。
