Vendor-specific HRP antibody conjugate differences lead to unexpected immunoassay reagent interactions.

IF 1.8 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Bioanalysis Pub Date : 2026-02-01 Epub Date: 2026-04-15 DOI:10.1080/17576180.2026.2658612

Nancy Yu, Justin Low, Frank Macchi, Richard Vandlen, James Zanghi, Benjamin T Andrews

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引用次数: 0

Abstract

Aim: An interaction observed in an anti-drug antibody (ADA) bridging ELISA between the drug and the anti-digoxin (aDIG) conjugated horseradish peroxidase (HRP) led to increased assay background across several commercial aDIG-HRP sources. Understanding the source of this interaction will help inform reagent qualities to be considered in production, evaluation, and selection for bioanalytical assays.

Methods and materials: This work evaluates commercial sources of aDIG-HRP through bridging ELISA, binding ELISA, SPR, and chromatography to determine what characteristics of aDIG-HRP lead to the interaction observed in a bridging ELISA.

Results: Three of four commercial aDIG-HRP sources evaluated exhibit drug binding and size heterogeneity with large (>250 kDa) species. SE-UHPLC of one source localizes this binding capability to the >6 MDa species. Under typical HRP conjugation conditions, HRP can conjugate to itself, and these larger species can bind to drug.

Conclusion: The conjugation of HRP to aDIG may lead to large species linked to HRP self-conjugation that can interfere with bioassays if not carefully controlled. Therefore, it is important to evaluate commercial reagents for consistency, availability, and critical quality when incorporating a new lot/vendor for better understanding of unexpected assay performance and effectively support reagent and assay life cycle management.

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供应商特异性HRP抗体偶联差异导致意想不到的免疫测定试剂相互作用。

目的：在抗药物抗体（ADA）桥接ELISA中观察到药物与抗地高辛（aDIG）偶联的辣根过氧化物酶（HRP）之间的相互作用，导致几种商业aDIG-HRP来源的测定背景增加。了解这种相互作用的来源将有助于告知在生产、评价和生物分析试验选择中要考虑的试剂质量。方法和材料：本工作通过桥接ELISA、结合ELISA、SPR和色谱法评估aDIG-HRP的商业来源，以确定aDIG-HRP的哪些特征导致桥接ELISA中观察到的相互作用。结果：在评估的四种商业aDIG-HRP来源中，有三种表现出药物结合和大（bb0 250 kDa）物种的大小异质性。一种来源的SE-UHPLC将这种结合能力定位于bbb60mda种。在典型的HRP偶联条件下，HRP可以与自身偶联，这些较大的物种可以与药物结合。结论：HRP与aDIG的偶联可能导致大量与HRP自偶联的物种，如果不小心控制，可能会干扰生物测定。因此，在合并新批次/供应商时，评估商业试剂的一致性、可用性和关键质量是很重要的，以便更好地了解意外的分析性能，并有效地支持试剂和分析生命周期管理。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Bioanalysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

3.30

自引率

16.70%

发文量

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.