Sprinkling in extra validation for high-value PTMs and therapeutic Abs with MILKSHAKE Western blots and Sundae ELISAs

IF 4.5 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

New biotechnology Pub Date : 2025-06-06 DOI:10.1016/j.nbt.2025.05.007

Qiana Mendez , Srinivas S. Thota , Jason J. Oliveira , Hannah J. Crary , Suchetana Saha , Kezzia S. Jones , Lourdes Perez , Felicity Acca , Cassandra D. Chapados , Holland A. Driscoll , Xiaofeng Li , Gregory R. Mirando , Mikhail A. Kostylev , Erik C. Gunther , Brian K. Kay , Michael P. Weiner , Mary R. Ferguson

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引用次数: 0

Abstract

Thoroughly validated antibodies (Abs) are crucial for the generation of meaningful scientific data. Abs for post translationally modified (PTM) protein targets in particular pose added validation challenges. The MILKSHAKE method employs surrogate proteins which are either modified or non-modified at a specific site. Western blot is used to observe the binding of PTM Abs to the surrogate proteins, indicating the specificity of the PTM Ab under test. In this study, we expand the utility of MILKSHAKE by validating acetyl and methyl specific Abs and by introducing another surrogate protein antigen based on cellulose binding domain (CBD) to evaluate Abs in a single western blot lane. This study also explores the use of ‘Sundae’ surrogate protein ELISA specifically for therapeutic Ab evaluation.

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用MILKSHAKE Western blots和Sundae elisa对高价值PTMs和治疗性抗体进行额外验证。

彻底验证的抗体（Abs）对于产生有意义的科学数据至关重要。特别是针对翻译后修饰（PTM）蛋白靶标的抗体带来了额外的验证挑战。奶昔法采用替代蛋白，在特定位点进行修饰或未修饰。Western blot观察PTM抗体与替代蛋白的结合情况，表明待测PTM抗体的特异性。在本研究中，我们通过验证乙酰基和甲基特异性抗体，并引入另一种基于纤维素结合域（CBD）的替代蛋白抗原，在单个western blot通道中评估抗体，扩大了MILKSHAKE的实用性。本研究还探讨了使用“Sundae”替代蛋白ELISA特异性用于治疗性Ab评估。

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

New biotechnology 生物-生化研究方法

CiteScore

11.40

自引率

1.90%

发文量

审稿时长

1 months

期刊介绍： New Biotechnology is the official journal of the European Federation of Biotechnology (EFB) and is published bimonthly. It covers both the science of biotechnology and its surrounding political, business and financial milieu. The journal publishes peer-reviewed basic research papers, authoritative reviews, feature articles and opinions in all areas of biotechnology. It reflects the full diversity of current biotechnology science, particularly those advances in research and practice that open opportunities for exploitation of knowledge, commercially or otherwise, together with news, discussion and comment on broader issues of general interest and concern. The outlook is fully international. The scope of the journal includes the research, industrial and commercial aspects of biotechnology, in areas such as: Healthcare and Pharmaceuticals; Food and Agriculture; Biofuels; Genetic Engineering and Molecular Biology; Genomics and Synthetic Biology; Nanotechnology; Environment and Biodiversity; Biocatalysis; Bioremediation; Process engineering.