A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry.
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.
Su
Jui-Lan
JL
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
Fornwald
Jim
J
Rivers
Philip
P
Goldsworthy
Susan
S
Looney
Noeleen A
NA
Hanvey
Jeff
J
Plumpton
Chris
C
Parham
Janet
J
Romanos
Michael
M
Kost
Thomas A
TA
Kull
Frederick C
FC
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
D000906:Antibodies; D007202:Indicators and Reagents; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins; C083929:neuropeptide FF receptor