TY - JOUR
T1 - High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics
AU - Lemos Duarte, Mariana
AU - Trimbake, Nikita A.
AU - Gupta, Achla
AU - Tumanut, Christine
AU - Fan, Xiaomin
AU - Woods, Catherine
AU - Ram, Akila
AU - Gomes, Ivone
AU - Bobeck, Erin N.
AU - Schechtman, Deborah
AU - Devi, Lakshmi A.
N1 - Funding Information:
We acknowledge Emily Chao, Kenix Vo, Jason Higa M.S., and Donghui Li, M.S. of AvantGen for their technical contributions to the screening, sequencing and antibody clone purification of all the antibody clones. We would like to thank Dr. Daniel Wacker, Dr. Michael J. Capper, Jeshua de Tenorio, Gregory Zilberg, and Audrey L. Warren for the technical support in the opioid receptor purification and its assembly into nanodiscs. We would like to thank Chenge Liu and Andrei Jeltyi for their help with the western blots. We would like to thank Alan Stern for the technical assistance using InCell microscope and Seshat Mack for critical reading of the manuscript. We would like to thank Joel Glovier for authorizing the use of the Erlenmeyer drawing in Fig. 2. This work was supported by NIH grants, 1R44DA35531 (to X.F.) and DA008863 and NS026880 (to L.A.D.), and Yale/NIDA Neuroproteomics Center Grant 5P30DA018343 (PI, Marina R. Picciotto).
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85101414373&partnerID=8YFLogxK
U2 - 10.1038/s42003-021-01744-8
DO - 10.1038/s42003-021-01744-8
M3 - Article
C2 - 33619305
AN - SCOPUS:85101414373
SN - 2399-3642
VL - 4
JO - Communications Biology
JF - Communications Biology
IS - 1
M1 - 238
ER -