Bioanalysis, Immunogenicity & Biomarkers, GlaxoSmithKline R&D, 1250 S Collegeville Road, Collegeville, PA 19426, USA.
Bioanalysis. 2022 Jan;14(1):19-28. doi: 10.4155/bio-2021-0197. Epub 2021 Nov 23.
Ruthenium-labeled antibodies are commonly used detection reagents in bioanalysis assays and must be characterized to ensure quality. The aim of this work was to develop a method to determine the concentration and incorporation ratio (the degree of labeling [DOL]) of ruthenium-labeled antibodies by UV/VIS spectroscopy. Free SULFO-TAG compound was scanned using UV/VIS and showed an absorbance peak at 292 nm. In contrast, antibodies demonstrate UV absorbance at 280 nm. After experimentally determining the extinction coefficients at 280 and 292 nm of free ruthenium and antibody, we generated a formula based on the Beer-Lambert law that calculates both concentration and DOL of these ruthenium-labeled antibodies. The concentration and DOL values determined by our method were comparable to those determined from bicinchoninic acid and LC/MS for the same reagents. This method creates a faster and more accessible reagent characterization process that uses far less reagent than the more traditional alternatives.
钌标记的抗体通常是生物分析检测中常用的检测试剂，必须对其进行特性鉴定以确保质量。本工作旨在开发一种通过紫外/可见分光光度法测定钌标记抗体浓度和结合比（标记程度[DOL]）的方法。游离的 SULFO-TAG 化合物经紫外/可见扫描，在 292nm 处有吸收峰，而抗体在 280nm 处有紫外吸收。在实验确定游离钌和抗体在 280nm 和 292nm 处的消光系数后，我们基于朗伯-比尔定律生成了一个公式，可计算这些钌标记抗体的浓度和 DOL。本方法测定的浓度和 DOL 值与同批试剂的比色法和 LC/MS 法测定的结果相当。该方法创建了一种更快、更易于操作的试剂特性鉴定方法，与传统方法相比，该方法所需的试剂更少。
