Orlov Alexey V, Malkerov Juri A, Novichikhin Denis O, Nikitin Alexei I, Sorokin Gennadiy M, Antopolsky Maxim L, Znoyko Sergey L
Prokhorov General Physics Institute of the Russian Academy of Sciences, Moscow, Russia.
National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 31 Kashirskoe shosse, 115409 Moscow, Russia.
MethodsX. 2022 Nov 8;9:101911. doi: 10.1016/j.mex.2022.101911. eCollection 2022.
Development of rapid and sensitive immunoassays is a task of great importance in a variety of fields ranging from clinical practice and urgent diagnostics to food quality control and environmental monitoring. High attention of researches is paid to methods of screening, selection, and kinetic characterization of antibodies that enable fast, specific, and effective formation of immunocomplexes. Herein, we present a method for direct investigation of kinetics of immunoreagents during developments of express high sensitive lateral flow assays. As model biomolecules to be detected, the following substances were tested: ochratoxin A (OTA), which is one of the most dangerous mycotoxins naturally present in many vegetable raw materials; and heart fatty acids binding protein (hFABP), which is a cardiac marker used in differential diagnosis of acute myocardial infarction. The kinetic constants of association (k) and dissociation (k) with monoclonal antibodies are determined along with the corresponding equilibrium constants (K and K). The obtained values are as follows: for the anti-OTA antibodies - k = 4.5410 Ms; k = 3.3210 s; K = 1.3710 M; K = 7.3110 M; and for the anti-hFABP antibodies - k = 7.2810 Ms; k = 1.9710 s; K = 3.7010 M; K = 2.7010 M. The proposed method can be employed in combination with the immunochromatographic assays based on magnetic biolabels.•Investigation of immunoreagent kinetics for development of express high sensitive lateral flow assays•Kinetic characterization of monoclonal antibodies against OTA and hFABP for their rapid and sensitive detection•Both kinetic and equilibrium constants of association and dissociation are determined.
在从临床实践和紧急诊断到食品质量控制和环境监测等各个领域，开发快速灵敏的免疫测定法是一项极为重要的任务。研究高度关注抗体的筛选、选择和动力学表征方法，这些方法能够实现免疫复合物的快速、特异性和有效形成。在此，我们提出一种在开发快速高灵敏侧向流动分析时直接研究免疫试剂动力学的方法。作为待检测的模型生物分子，测试了以下物质：赭曲霉毒素A（OTA），它是许多蔬菜原料中天然存在的最危险的霉菌毒素之一；以及心脏脂肪酸结合蛋白（hFABP），它是用于急性心肌梗死鉴别诊断的心脏标志物。测定了与单克隆抗体的结合动力学常数（k）和解离动力学常数（k）以及相应的平衡常数（K和K）。获得的值如下：抗OTA抗体 - k = 4.54×10⁶ M⁻¹s⁻¹；k = 3.32×10⁻³ s⁻¹；K = 1.37×10⁶ M⁻¹；K = 7.31×10⁻⁷ M；抗hFABP抗体 - k = 7.28×10⁶ M⁻¹s⁻¹；k = 1.97×10⁻³ s⁻¹；K = 3.70×10⁶ M⁻¹；K = 2.70×10⁻⁷ M。所提出的方法可与基于磁性生物标记的免疫层析测定法结合使用。•用于开发快速高灵敏侧向流动分析的免疫试剂动力学研究•针对OTA和hFABP的单克隆抗体的动力学表征以实现其快速灵敏检测•测定了结合和解离的动力学及平衡常数
