抗体
免疫分析
多克隆抗体
融合蛋白
蛋白质G
分析物
化学
抗原
分子生物学
核蛋白
蛋白质A
免疫球蛋白G
病毒学
生物
色谱法
病毒
生物化学
重组DNA
免疫学
基因
作者
Suman Nandy,Mary Crum,Katherine Wasden,Ulrich Strych,Atul Goyal,Vijay Maranholkar,William Mo,Binh Vu,Katerina Kourentzi,Richard C. Willson
标识
DOI:10.1016/j.ab.2022.114929
摘要
Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immunoassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).
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