Method for the validation by Western analysis of affinity reagents against post-translationally modified proteins.

通过蛋白质印迹分析验证亲和试剂针对翻译后修饰蛋白质的方法。

• 批准号: 10819809
• 负责人: Michael P Weiner
• 金额: $ 112.2万
• 依托单位: ABBRATECH, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10819809
• 关键词: Acetylation; Address; Affect; Affinity; Antibodies; Antibody Binding Sites; Antibody Specificity; Area; Benchmarking; Binding; Biological Assay; Biomedical Research; Biotechnology; Catalogs; Collaborations; Communities; Custom; Data; Detection; Development; Disease; Educational workshop; Ensure; Epitopes; Flow Cytometry; Future; Genetic Polymorphism; Goals; Knowledge; Malignant Neoplasms; Mammalian Cell; Manuals; Marketing; Measures; Methods; Methylation; Modeling; Molecular Biology; Names; Pharmacologic Substance; Phase; Phosphorylation; Phosphorylation Site; Post-Translational Protein Processing; Protein Glycosylation; Proteins; Proteome; Proteomics; Protocols documentation; Publishing; Reagent; Reproducibility; Research; Research Personnel; Sensitivity and Specificity; Services; Site; Small Business Innovation Research Grant; Specificity; Techniques; Technology; Therapeutic antibodies; United States National Institutes of Health; Validation; Work; central database; commercialization; cross reactivity; glycosylation; improved; industry partner; innovation; insight; ipilimumab; shared database; sharing platform; success; symposium; technology validation; tool

ABSTRACT Antibodies (Abs) are commonly employed in biomedical research to identify and quantify target proteins. However, the specificity, sensitivity and reliability of these Abs can be variable, which is a significant concern in the scientific community. The use of an incorrect Ab can result in misleading or inaccurate results, impeding progress in various areas of biomedical research. In Phase I of this project, we developed tools to validate the specificity of commercial Abs used to detect phosphorylated sites in proteins in a method we developed and published that we named MILKSHAKE. We will present some of these results from this Phase I effort, which demonstrates the critical need to address this concern. This proposed SBIR Phase II project aims to both extend and commercialize the Phase I effort. We will continue the Ab validation work initiated in Phase I by extending the analysis to other types of post-translational modifications (PTMs) of proteins in Western analysis and flow cytometry. This Phase II project also seeks to improve and scale this pipeline further, and to develop new methods for validating Abs against glycosylated proteins, which are often dynamic and challenging to detect using traditional techniques. We will also employ an innovative tool for measuring Ab promiscuity using a technology we term ‘Sundae’ to better investigate the specificity of the Ab paratope. Sundae will be demonstrated for two therapeutic Abs. The Phase II project has several key objectives, including the development of new Ab-specific assays and the establishment of a centralized database for sharing validated Ab data. The project team comprises experts in Ab validation, proteomics, and molecular biology, and will collaborate closely with our current and future industry partners and academic researchers to ensure our success.

抽象的 抗体（ABS）通常用于生物医学研究中，以识别和量化靶标 蛋白质。但是，这些ABS的特异性，灵敏度和可靠性可能是可变的，这是 科学界的重大关注。使用不正确的AB会导致误导 或结果不准确，阻碍了生物医学研究的各个领域的进步。在第一阶段 项目，我们开发了验证用于检测商业ABS的特异性的工具 蛋白质中的磷酸化位点我们开发和发表了我们命名的方法 奶昔。我们将在此阶段I努力中提出其中的一些结果，这证明了 解决这一问题的迫切需要。该拟议的SBIR II期项目旨在扩展和 商业化第一阶段的努力。我们将继续由AB验证工作在第一阶段发起的工作 将分析扩展到其他类型的蛋白质的翻译后修饰（PTM） 西方分析和流式细胞仪。该第二阶段项目还旨在改善和扩展此事 进一步的管道，并开发针对糖基化蛋白验证ABS的新方法，该方法 我们还将采用 使用技术“圣代”的技术来衡量AB滥交的创新工具以更好 研究AB副群的特异性。圣代表将用于两个治疗性ABS。 第二阶段项目具有多个关键目标，包括开发新的AB特定目标 测定和建立用于共享已验证的AB数据的集中数据库。项目 团队建立AB验证，蛋白质组学和分子生物学方面的专家，并将合作 与当前和未来的行业合作伙伴和学术研究人员紧密相关，以确保我们的 成功。

项目成果

MILKSHAKE Western blot and Sundae ELISA: We all scream for better antibody validation.

MILKSHAKE 蛋白质印迹和圣代 ELISA：我们都渴望更好的抗体验证。

• DOI: 10.1016/j.jim.2023.113540
• 发表时间: 2023
• 期刊: Journal of immunological methods
• 影响因子: 2.2
• 作者: Mendez,Qiana;Driscoll,HollandA;Mirando,GregoryR;Acca,Felicity;Chapados,CassandraD;Jones,KezziaS;Weiner,Michael;Li,Xiaofeng;Ferguson,MaryR
• 通讯作者: Ferguson,MaryR

MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

• DOI: 10.2144/btn-2021-0078
• 发表时间: 2022-01
• 期刊: BioTechniques
• 影响因子: 2.7

Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols.

使用奶昔和圣代方案验证和测定抗体生物活性。

• DOI: 10.1007/978-1-0716-3381-6_24
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Ferguson,MaryR;Mendez,QianaM;Acca,FelicityE;Chapados,CassandraD;Driscoll,HollandA;Jones,KezziaS;Mirando,Gregory;Weiner,MichaelP;Li,Xiaofeng
• 通讯作者: Li,Xiaofeng