High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics

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.


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No statistical methods were used to predetermine sample size. At the high-throughput microscopy analysis, 1000 -10000 cells were acquired in each biological replicate, allowing data reproducibility.
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All attempts at replication were successful, the experimental replicates were performed in triplicates/quaduplicates, in each biological replicate experiment. For the high-throughput microscopy analysis, were acquired at least 10 fields for each condition in each biological replicate. Total 2-4 biological replicates on these experiments.
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On this paper we performed the validation of all the antibodies presented using techniques such as western blot, ELISA, immunofluorescence. We generated knockout cell lines to