A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry

J Immunol Methods. 2004 Aug;291(1-2):123-35. doi: 10.1016/j.jim.2004.05.006.

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.

MeSH terms

  • Antibodies / chemistry
  • Antibodies / genetics
  • Antibodies / immunology*
  • Baculoviridae / genetics
  • Baculoviridae / physiology
  • Cell Line
  • Enzyme-Linked Immunosorbent Assay
  • Fluorescence
  • Humans
  • Immunohistochemistry / methods*
  • Indicators and Reagents
  • Receptors, G-Protein-Coupled / immunology*
  • Receptors, Neuropeptide / genetics
  • Receptors, Neuropeptide / immunology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sensitivity and Specificity
  • Time Factors
  • Titrimetry
  • Transduction, Genetic

Substances

  • Antibodies
  • Indicators and Reagents
  • Receptors, G-Protein-Coupled
  • Receptors, Neuropeptide
  • Recombinant Proteins
  • neuropeptide FF receptor