Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the ‘opioid epidemic.’ We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.

抗体是检查信号转导途径的强大工具。在这里，我们提出了一种整合多种最先进方法来生产、验证和利用抗体的策略。我们专注于尚未研究的突触蛋白，生成了 137 种重组抗体。我们使用来自免疫兔 B 细胞的酵母展示抗体文库，然后在严格条件下进行 FACS 分选，以鉴定高亲和力抗体。这些抗体通过高通量功能筛选和基因组编辑进行了验证。接下来，我们探索了单细胞信号传导的时间动态。一组针对阿片类药物受体的抗体被用来检查阿片类药物治疗的效果，这些药物在“阿片类药物流行病”的恶化中发挥了核心作用。我们发现吗啡和芬太尼表现出受体磷酸化的差异时间动力学。总之，高通量方法可以识别深入了解阿片类药物信号传导时间动态所需的基于抗体的工具。