定量检测PD-1单克隆抗体ELISA法的建立

Development and application of ELISA method for quantitative detection of PD-1 monoclonal antibody

目的建立一种高特异、高灵敏、新型的定量检测食蟹猴血清中PD-1单克隆抗体质量浓度的ELISA方法。方法用重组人PD-1作为抗原包被到96孔板上,加入系列浓度标准品(PD-1单克隆抗体)和稀释的食蟹猴血清,再加入稀释的猴血清吸附的羊抗人Ig G-HRP(二抗),二抗与结合到抗原上的PD-1单克隆抗体特异性结合,加入底物显色剂,用硫酸终止,在450 nm下读数。结果在40~0.625 ng/ml范围内,PD-1单克隆抗体质量浓度的对数值与吸光值呈S形曲线,方法灵敏度为0.625 ng/ml,TNF-α、r HSA、PEG-GH和受试品PD-1单克隆抗体均无交叉反应,方法的回收率为94.5%~98.0%,板内精密度波动在-4.1%^-0.1%,板间精密度波动在-5.0%^-1.0%。结论本方法符合新生物制品临床前药代动力学研究指导原则的要求,可用于食蟹猴体内PD-1单克隆抗体的定量检测。

This study was performed to develop a specific and sensitive ELISA method for the quantification of PD-1 monoclonal antibody in eynomolgus monkey serum. Recombinant human PD-1 was coated onto 96-well plates, and then standard preparation of PD-1 monoclonal antibody at different concentrations and diluted cynomolgus serum were added. When PD-1 monoclonal antibody in serum was bound to the recombinant human PD-1 specifically, goat anti-human IgG-HRP （secondary antibody） diluted by monkey serum was added, which then bound to PD-1 monoclonal antibody. Then substrate reagent was added and sulfuric acid was used for quenching, and the optical density at 450 nm was detected. The standard curve of the constructed method was developed with a wide dynamic range of concentrations from 0.625 to 40 ng/ml. The lowest quantification of this assay was 0.625 ng/ml. The specificity assay indicated that PD-1 antibody in samples had no cross-reaction with TNF-α, rHSA, and PEG-GH; the recovery of the method was 94.5%-98.0%, and the precision degree of the intraand inter-assay were varied between -4.1%- -0.1% and -5.0%- -1.0%, respectively. Taken together, this method is consistent with the requirements of the new research guidelines of biological preclinical pharmacokinetics, with highly sensitive, precise, specific, and reproducibility over a wide dynamic range of concentrations, which is proven to be a feasible quantitative method for detection of PD-1 monoclonal antibody in the serum of cynomolgus monkeys.