High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics.
Commun Biol
; 4(1): 238, 2021 02 22.
Article
em En
| MEDLINE
| ID: mdl-33619305
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Sinapses
/
Proteína Quinase C
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Receptores Opioides mu
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Ensaios de Triagem em Larga Escala
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Anticorpos
/
Especificidade de Anticorpos
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limite:
Animals
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Humans
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Male
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article