Multiplexed assays to determine the selectivity of anti-GPCR antibodies

Description of study and method This dataset contains measurement values from a multiplexed screen of antibodies (Abs) to determine their selective recognition of G protein-coupled receptors (GPCRs). Therapeutic antibodies are being developed to modulate GPCR function. However, validating the selectivity of anti-GPCR Abs is challenging due to sequence similarities of individual receptors within GPCR subfamilies. Here we present data from multiplexed immunoassay to test >400 anti-GPCR Abs from the Human Protein Atlas targeting a customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. The GPCRs were conjugated with 1D4 and FLAG epitope tags (common protein tags for Ab binding), were overexpressed in Expi293F cells and solubilized for the assay. The Abs were tested using the suspension bead array (SBA) technology from Luminex. The technology employs color-coded beads with Abs that bind the target (for capture) and fluorescently labelled Abs that bind the 1D4 epitope tag (for detection). The resulting output data are the median fluorescence intensity (MFI) values for each sample and Ab. MFI represents a relative measurement that allows for comparison within Abs but not between. The dataset also contains Z-scores and robust Z-scores for visualization and to provide a similar scale for all Abs. For Ab validation, Abs targeting GPCRs were used for capture. To verify that GPCRs were expressed at all, Abs targeting the FLAG tag were used for capture. Each anti-GPCR Ab was evaluated against its target GPCR and phenotypically closely related GPCRs by using a population density-based threshold to map the on-target and off-target binding of the antibody. The files contain the following data and information abval_dat.csv: Sample information and Luminex measurements (MFI, Z-scores and robust Z-scores) for samples used in the antibody validation screen (GPCR capture, 1D4 detection). expr_dat.csv: Sample information and Luminex measurements for lysates used to evaluate GPCR expression (FLAG capture, 1D4 detection). prot.csv: Information about antibodies used in the study. ReadMe: Details about the files and description of columns研究与方法说明 本数据集包含来自多重抗体筛选实验的检测数据，用于评估抗体（Antibodies，下文简称Abs）对G蛋白偶联受体（G protein-coupled receptors, GPCRs）的选择性识别能力。当前学界正开发治疗性抗体以调控GPCR的功能。然而，由于GPCR亚家族内各受体的序列相似度较高，验证抗GPCR抗体的选择性颇具挑战。 本研究通过多重免疫测定实验，对来自人类蛋白质图谱（Human Protein Atlas）的逾400种抗GPCR抗体进行检测，这些抗体靶向涵盖所有GPCR亚家族的215个已表达并可溶的GPCR定制化文库。本次实验中，所有GPCR均偶联了1D4与FLAG表位标签（二者为常用于抗体结合的蛋白标签），并在Expi293F细胞中过表达，随后被提取溶解以用于检测。 本次检测采用Luminex公司的悬液微珠阵列（Suspension Bead Array, SBA）技术。该技术依托带颜色编码的微珠：其中包被的抗体用于捕获靶标，而荧光标记的抗体则结合1D4表位标签以实现检测。最终输出的数据为每个样本与抗体对应的中位荧光强度（Median Fluorescence Intensity, MFI）值。MFI是一种相对定量指标，仅可用于同一种抗体的不同样本间比较，无法用于不同抗体间的比对。 本数据集同时包含Z分数与稳健Z分数，用于数据可视化，并为所有抗体的检测结果提供统一的量化尺度。在抗体验证环节，以靶向GPCR的抗体作为捕获抗体；而为验证GPCR是否成功表达，则采用靶向FLAG标签的抗体作为捕获抗体。本研究基于群体密度阈值，针对每种抗GPCR抗体，分别对其靶标GPCR以及表型上密切相关的GPCR进行评估，以明确该抗体的靶向结合与非靶向结合模式。 本数据集包含以下文件与相关数据信息： abval_dat.csv：抗体筛选验证实验的样本信息与Luminex检测数据（含MFI、Z分数及稳健Z分数），该实验采用GPCR捕获、1D4检测的实验方案。 expr_dat.csv：用于评估GPCR表达的裂解液样本信息与Luminex检测数据，该实验采用FLAG捕获、1D4检测的实验方案。 prot.csv：本研究中使用的抗体相关信息。 ReadMe：各文件的详细说明与数据列字段解释。