Abucayon Erwin G, Whalen Connor, Torres Oscar B, Duval Alexander J, Sulima Agnieszka, Antoline Joshua F G, Oertel Therese, Barrientos Rodell C, Jacobson Arthur E, Rice Kenner C, Matyas Gary R
Laboratory of Adjuvant and Antigen Research, U.S. Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910, United States.
Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, Maryland 20817, United States.
ACS Omega. 2022 Jul 19;7(30):26812-26823. doi: 10.1021/acsomega.2c03237. eCollection 2022 Aug 2.
The quantitation of the available antibody binding-site concentration of polyclonal antibodies in serum is critical in defining the efficacy of vaccines against substances of abuse. We have conceptualized an equilibrium dialysis (ED)-based approach coupled with fluorimetry (ED-fluorimetry) to measure the antibody binding-site concentration to the ligand in an aqueous environment. The measured binding-site concentrations in monoclonal antibody (mAb) and sera samples from TT-6-AmHap-immunized rats by ED-fluorimetry are in agreement with those determined by a more established equilibrium dialysis coupled with ultraperformance liquid chromatography tandem mass spectrometry (ED-UPLC-MS/MS). Importantly, we have shown that the measured antibody binding-site concentrations to the ligand by ED-fluorimetry were not influenced by the sample serum matrix; thus, this method is valid for determining the binding-site concentration of polyclonal antibodies in sera samples. Further, we have demonstrated that under appropriate analytical conditions, this method resolved the total binding-site concentrations on a nanomolar scale with good accuracy and repeatability within the microliter sample volumes. This simple, rapid, and sample preparation-free approach has the potential to reliably perform quantitative antibody binding-site screening in serum and other more complex biological fluids.
血清中多克隆抗体可用抗体结合位点浓度的定量对于确定抗滥用物质疫苗的疗效至关重要。我们构思了一种基于平衡透析（ED）并结合荧光法（ED-荧光法）的方法，以测量水性环境中抗体与配体的结合位点浓度。通过ED-荧光法测得的来自经TT-6-AmHap免疫的大鼠的单克隆抗体（mAb）和血清样品中的结合位点浓度，与通过更成熟的平衡透析结合超高效液相色谱串联质谱法（ED-UPLC-MS/MS）测定的结果一致。重要的是，我们已经表明，通过ED-荧光法测得的抗体与配体的结合位点浓度不受样品血清基质的影响；因此，该方法对于测定血清样品中多克隆抗体的结合位点浓度是有效的。此外，我们已经证明，在适当的分析条件下，该方法在纳摩尔尺度上解析了总结合位点浓度，在微升样品体积内具有良好的准确性和重复性。这种简单、快速且无需样品制备的方法有潜力在血清和其他更复杂的生物流体中可靠地进行定量抗体结合位点筛选。
