Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, Interdisciplinary Fabry Center Muenster (IFAZ), University Hospital Muenster, 48149 Muenster, Germany.
Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, 48149 Muenster, Germany.
Int J Mol Sci. 2021 Mar 6;22(5):2680. doi: 10.3390/ijms22052680.
Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant α-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-α, agalsidase-β and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r = 0.9063 and r = 0.8952, both < 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-α, agalsidase-β and Moss-AGAL (KD: 1.94 ± 0.11 µM, 2.46 ± 0.21 µM, and 1.33 ± 0.09 µM, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-α, agalsidase-β and Moss-AGAL.
男性法布里病 (FD) 患者对用于酶替代疗法的重组α-半乳糖苷酶 A (AGAL) 形成抗体的风险很高。由于疾病进展迅速，因此非常需要确定有风险的患者。然而，目前不存在用于测定抗药物抗体 (ADA) 的合适参考标准和标准化方案。在这里，我们生成了一个全面的患者衍生抗体混合物作为参考，允许通过 ELISA 从单个血样中定量测定抗体滴度。将 22 名 FD 男性患者的血清样本与针对 AGAL 的 ADA 混合，并通过免疫吸附进行纯化。通过石英晶体微天平耗散监测 (QCM-D) 测量 ADA 对 agalsidase-α、agalsidase-β 和 Moss-AGAL 的亲和力。AGAL 特异性免疫吸附产生了一种多克隆 ADA 混合物，显示出浓度依赖性结合和对 AGAL 的抑制。原始血清和纯化总 IgG 的滴度 (r = 0.9063 和 r = 0.8952，均 <0.0001) 与 ADA 的个体抑制能力相关。QCM-D 测量表明，参考抗体对 agalsidase-α、agalsidase-β 和 Moss-AGAL 的亲和力相当 (KD：1.94 ± 0.11 µM、2.46 ± 0.21 µM 和 1.33 ± 0.09 µM)。该参考抗体允许基于 ELISA 的 ADA 滴度测定和绝对浓度的定量。此外，FD 患者的 ADA 对 agalsidase-α、agalsidase-β 和 Moss-AGAL 的亲和力相当。
