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Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
Published on: September 13, 2013
A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor
Jui-Lan Su1, Jim Fornwald, Philip Rivers
1Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline Research and Development, Five Moore Drive, Research Triangle Park, NC 27709, USA. jui-lan.c.su@gsk.com
Insights
A new cell-based time-resolved fluorescence (celTRF) immunoassay effectively pre-screens antibodies for G protein-coupled receptor (GPCR) research. This assay accurately predicts antibody suitability for immunohistochemistry (IHC), unlike traditional ELISA methods.
Area of Science:
- Biochemistry and Molecular Biology
- Immunology
- Cell Biology
Background:
- G protein-coupled receptors (GPCRs) are crucial drug targets, necessitating high-quality antibodies for research.
- Traditional antibody screening methods like ELISA often fail to predict efficacy in downstream applications such as immunohistochemistry (IHC).
- There is a need for reliable in vitro assays to pre-screen antibody suitability for IHC.
Purpose of the Study:
- To develop and validate a cell-based time-resolved fluorescence (celTRF) immunoassay for pre-screening antibodies against GPCR peptides.
- To assess the celTRF assay's ability to predict antibody suitability for immunohistochemistry (IHC).
- To compare the predictive power of celTRF with traditional ELISA for IHC applications.
Main Methods:
- GPCRs were expressed in Saos-2 cells using recombinant baculoviruses, creating a "screening lawn" fixed and permeabilized similarly to IHC tissue.
- A dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) using Eu-labelled goat anti-rabbit IgG was employed for celTRF.
- Over 150 anti-peptide reagents for 27 GPCRs were characterized, with manual and automated (Biomek FX, Sami) assay performance evaluated.
Main Results:
- The celTRF assay demonstrated a broad dynamic range and high specificity and potency for antibody characterization.
- All celTRF-positive antibodies were suitable for IHC, while ELISA alone did not reliably predict IHC utility.
- A strong correlation between celTRF potency and IHC utility was observed, exemplified by anti-neuropeptide FF receptor 1 (NPFF1) antibodies.
Conclusions:
- The celTRF immunoassay is the first automated in vitro assay for confident pre-selection of antibodies for IHC.
- celTRF offers superior prediction of antibody utility for IHC compared to ELISA.
- The celTRF assay is versatile and applicable to a wide range of GPCRs and other protein target classes.
Abstract:
A cell-based time-resolved fluorescence (celTRF) immunoassay is described for pre-screening antibodies to G protein-coupled receptor (GPCR) peptides that predicts suitability for immunohistochemistry (IHC). Rat GPCRs were expressed in Saos-2 human osteosarcoma cells via recombinant baculoviruses designed for mammalian cell expression, i.e., the transduced cells were used as a "screening lawn". The lawn was fixed and permeabilized similarly to IHC tissue. The celTRF, a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), employed Eu-labelled goat anti-rabbit IgG. It exhibited a broad dynamic range upon which enzyme-linked immunosorbant assay (ELISA)-positive affinity-purified anti-peptide antibody reagents were examined for specificity and potency. Over 150 anti-peptide reagents to 27 GPCRs were characterized. All celTRF-positive antibodies were found to be suitable for IHC, whereas ELISA alone did not predict IHC utility. Examples are illustrated with five rabbit anti-neuropeptide FF receptor 1 (NPFF1) antibodies, where a strong correlation between celTRF potency and IHC utility was observed in both applications. In contrast, two high anti-peptide ELISA titer but celTRF-negative antibodies failed to recognize the NPFF1 receptor in IHC. The celTRF assay was performed manually and in an automated fashion, in our case, using a Biomek FX station and Sami scheduling software. The celTRF is the first in vitro automated assay that offers confident pre-selection of antibodies for IHC and the versatility to accommodate the rapid screening of large numbers of GPCRs. The celTRF is readily applicable to other protein target classes.
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