title: A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry.
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.
authors.affiliation: Department 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
authors.forename: Jui-Lan
authors.initials: JL
authors.lastname: Su
authors.forename: Jim
authors.initials: J
authors.lastname: Fornwald
authors.forename: Philip
authors.initials: P
authors.lastname: Rivers
authors.forename: Susan
authors.initials: S
authors.lastname: Goldsworthy
authors.forename: Noeleen A
authors.initials: NA
authors.lastname: Looney
authors.forename: Jeff
authors.initials: J
authors.lastname: Hanvey
authors.forename: Chris
authors.initials: C
authors.lastname: Plumpton
authors.forename: Janet
authors.initials: J
authors.lastname: Parham
authors.forename: Michael
authors.initials: M
authors.lastname: Romanos
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kost
authors.forename: Frederick C
authors.initials: FC
authors.lastname: Kull
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007202:Indicators and Reagents; D043562:Receptors, G-Protein-Coupled / Q000276:immunology*; D018013:Receptors, Neuropeptide / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors; D017186:Titrimetry; D014161:Transduction, Genetic
chemical_list: D000906:Antibodies; D007202:Indicators and Reagents; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins; C083929:neuropeptide FF receptor