Green fluorescent antibodies: novel in vitro tools.
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.
Casey
J L
JL
Department of Biochemistry and CRC for Diagnostic Technologies, La Trobe University, Bundoora, Victoria, 3083, Australia.
Coley
A M
AM
Tilley
L M
LM
Foley
M
M
D000818:Animals; D000918:Antibody Specificity; D019556:COS Cells; D002678:Chimera; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D057127:Single-Chain Antibodies; D014162:Transfection
D006514:Hepatitis B Surface Antigens; D007135:Immunoglobulin Variable Region; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C410726:scFV green fluorescent protein, recombinant; D049452:Green Fluorescent Proteins