磁性颗粒在快速测定镓标记的抗PD-L1 VHH抗体免疫反应性组分中的应用
Application of Magnetic Particles for Fast Determination of Immunoreactive Fraction of Ga-Labelled VHH Antibodies to PD-L1.
作者信息
Avrov K O, Shatik S V, Zaitsev V V, Al-Shehadat R I, Shashkova O A, Terekhina L A, Malakhov I S, Samoylovich M P
机构信息
Senior Researcher, Laboratory of Hybridome Technology; A.M. Granov Russian Research Center of Radiology and Surgical Technologies of the Ministry of Health of the Russian Federation, 70 Leningradskaya St., Saint Petersburg, Pesochniy Settlement, 197758, Russia.
Head of the Department of Cyclotron-Produced Radiopharmaceuticals; A.M. Granov Russian Research Center of Radiology and Surgical Technologies of the Ministry of Health of the Russian Federation, 70 Leningradskaya St., Saint Petersburg, Pesochniy Settlement, 197758, Russia.
出版信息
Sovrem Tekhnologii Med. 2023;15(3):26-33. doi: 10.17691/stm2023.15.3.03. Epub 2023 May 28.
UNLABELLED
Quantification of the immunoreactive fraction (IRF) of radioactive isotope-labeled antibodies or their fragments is necessary to assess the specific activity of radiopharmaceuticals. Traditionally, cells expressing the target molecules on their surface are used to determine IRF, but such analysis is time-consuming and has difficulties with standardization. was to develop a fast and reliable method for quantitative determination of IRF by Ga-labeled VHH antibodies to PD-L1 based on the use of magnetic particles coated with antigen molecules.
MATERIALS AND METHODS
Commercially available magnetic particles coated with protein A have been used in our study. The antigen conjugated with the Fc fragment (PD-L1-Fc) was immobilized on the particles. The IRF value of Ga radionuclide-labeled nanobodies (VHH) against PD-L1 (Ga-VHH-PD-L1) was determined using magnetic particles coated with antigen molecules and cells expressing the antigen on their surface. When VHH antibodies were conjugated to Ga radionuclide, protein molecules were modified using bifunctional chelating agents: tetraazacyclododecanetetraacetic acid (DOTA) or deferoxamine (DFO). The magnitude of IRF was defined as the ratio of radioactivity specifically bound to particles or cells to the total radioactivity added to the sample.
RESULTS
The specificity of the Ga-VHH-PD-L1 radioimmunoconjugate binding to the antigen-coated magnetic particles has been proved. Some special aspects, which should be taken into consideration when using this method, have been established. The comparison of the IRF estimates using the antigen-expressing cells and magnetic particles has not revealed any significant differences in the results obtained in our study. Nevertheless, the presented method based on magnetic particles with immobilized antigen molecules requires only 15 min to determine the radioimmunoconjugate IRF, which is of fundamental importance for the routine assessment of the specificity of radiopharmaceuticals containing short-lived isotopes.
未标记
对放射性同位素标记的抗体或其片段的免疫反应性分数（IRF）进行定量，对于评估放射性药物的比活度是必要的。传统上，使用在其表面表达靶分子的细胞来确定IRF，但这种分析耗时且难以标准化。本研究旨在开发一种基于使用包被有抗原分子的磁性颗粒，通过镓标记的抗PD-L1 VHH抗体快速可靠地定量测定IRF的方法。
材料与方法
我们的研究中使用了市售的包被有蛋白A的磁性颗粒。与Fc片段偶联的抗原（PD-L1-Fc）固定在颗粒上。使用包被有抗原分子的磁性颗粒和在其表面表达抗原的细胞，测定镓放射性核素标记的抗PD-L1纳米抗体（VHH）（镓-VHH-PD-L1）的IRF值。当VHH抗体与镓放射性核素偶联时，使用双功能螯合剂：四氮杂环十二烷四乙酸（DOTA）或去铁胺（DFO）修饰蛋白质分子。IRF的大小定义为与颗粒或细胞特异性结合的放射性与加入样品中的总放射性之比。
结果
已证明镓-VHH-PD-L1放射免疫缀合物与包被有抗原的磁性颗粒结合的特异性。已确定了使用该方法时应考虑的一些特殊方面。使用表达抗原的细胞和磁性颗粒对IRF估计值的比较未发现我们研究中获得的结果有任何显著差异。然而，所提出的基于固定有抗原分子的磁性颗粒的方法仅需15分钟即可确定放射免疫缀合物的IRF，这对于常规评估含有短寿命同位素的放射性药物的特异性至关重要。
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