A Homogeneous and Noncompetitive Immunoassay Based on the Enhanced Fluorescence Resonance Energy Transfer by Leucine Zipper Interaction

• Published in: Analytical chemistry (Washington) Vol. 74; no. 22; pp. 5786 - 5792
• Main Authors: Ohiro, Yoshiyuki, Arai, Ryoichi, Ueda, Hiroshi, Nagamune, Teruyuki
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-11-2002
• Subjects: Analytical chemistry; Chemistry; Energy Transfer; Enzyme-Linked Immunosorbent Assay; Exact sciences and technology; Fluorescence; Green Fluorescent Proteins; Humans; Immunoassay - methods; Leucine Zippers; Luminescent Proteins - chemistry; Miscellaneous; Plasmids - genetics; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Proteins - chemistry; Spectrometric and optical methods; Spectrometry, Fluorescence; Investigation method; Zinc finger structure; Green fluorescent protein; Sandwich structure; Fluorescence; Resonance energy; Leucine; Energy transfer; Fluorescence spectrometry; Immunological method
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac0203387

Fluorescence resonance energy transfer (FRET) between two GFP variants is a powerful technique to describe protein−protein interaction in a biological system. However, it has a limitation that the two variants tethered to the respective proteins have to be in sufficient proximity upon binding, which is often difficult to attain by simple N- or C-terminal fusions. Here we describe a novel method to significantly enhance FRET between GFP variant-tagged proteins with the use of leucine zippers. For the homogeneous sandwich immunoassay of a high molecular weight antigen human serum albumin (HSA), two separate single-chain Fvs recognizing distant epitopes of HSA were respectively fused with fluorescence donor ECFP or acceptor EYFP, and FRET between the two was analyzed by fluorescence spectrometry. Because these two proteins did not give any detectable FRET upon antigen addition, we tethered each protein with a leucine zipper motif (c-Jun or FosB) at the C-terminus to help the neighborhood of the GFP variants. Upon antigen addition, the new pairs showed significant antigen-dependent FRET. By exchanging the binding domains, the method will find a range of applications for the assay of other proteins and their interactions in vitro or in vivo.