Green fluorescent antibodies: novel in vitro tools

Protein Eng. 2000 Jun;13(6):445-52. doi: 10.1093/protein/13.6.445.


We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibody Specificity
  • COS Cells
  • Chimera
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Evaluation Studies as Topic
  • Fluorescence
  • Fluorescent Antibody Technique
  • Green Fluorescent Proteins
  • Hepatitis B Surface Antigens / biosynthesis
  • Hepatitis B Surface Antigens / genetics
  • Hepatitis B Surface Antigens / immunology
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / isolation & purification
  • Immunoglobulin Variable Region / metabolism*
  • Luminescent Proteins / analysis
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / genetics*
  • Luminescent Proteins / isolation & purification
  • Luminescent Proteins / metabolism*
  • Protein Binding / immunology
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism*
  • Single-Chain Antibodies
  • Transfection


  • Hepatitis B Surface Antigens
  • Immunoglobulin Variable Region
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Single-Chain Antibodies
  • scFV green fluorescent protein, recombinant
  • Green Fluorescent Proteins