Blake A Rowe, Katie Medina-Carle, Keguan Chen, Kimberly J Reese, Kenneth M McCarthy, Amy A Concannon, George R Gunn, Andrew P Gehman, Yong Jiang, Erik Meyer
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引用次数: 0
摘要
目的:重新开发一种中和抗体(NAb)检测方法,使其具有更强的药物耐受性、更大的动态范围以及在使用高水平阳性对照(PC)时具有更高的抑制率。材料与方法:早期的检测数据表明,捕获试剂(在人类细胞系中产生的药物 1)的典型生物素标记会阻碍其与 PC 或检测目标的结合,而且检测目标会超越 PC。我们以几种挑战比进行了有条不紊的生物素标记实验,并在检测靶标上添加了 Fc 连接器。结果与结论:通过在检测中添加酸解离步骤,对药物 1 进行非典型生物素标记,并改用含有 Fc 连接物的检测靶标来增加立体阻碍并降低其与药物 1 的结合亲和力,从而实现了更大的动态范围、更高的抑制率和更高的药物耐受性。
Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.
Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC).Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target.Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.
BioanalysisBIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL
CiteScore
3.30
自引率
16.70%
发文量
88
审稿时长
2 months
期刊介绍:
Reliable data obtained from selective, sensitive and reproducible analysis of xenobiotics and biotics in biological samples is a fundamental and crucial part of every successful drug development program. The same principles can also apply to many other areas of research such as forensic science, toxicology and sports doping testing.
The bioanalytical field incorporates sophisticated techniques linking sample preparation and advanced separations with MS and NMR detection systems, automation and robotics. Standards set by regulatory bodies regarding method development and validation increasingly define the boundaries between speed and quality.
Bioanalysis is a progressive discipline for which the future holds many exciting opportunities to further reduce sample volumes, analysis cost and environmental impact, as well as to improve sensitivity, specificity, accuracy, efficiency, assay throughput, data quality, data handling and processing.
The journal Bioanalysis focuses on the techniques and methods used for the detection or quantitative study of analytes in human or animal biological samples. Bioanalysis encourages the submission of articles describing forward-looking applications, including biosensors, microfluidics, miniaturized analytical devices, and new hyphenated and multi-dimensional techniques.
Bioanalysis delivers essential information in concise, at-a-glance article formats. Key advances in the field are reported and analyzed by international experts, providing an authoritative but accessible forum for the modern bioanalyst.