Application of Green Fluorescent Protein in Immunoassays

Green fluorescent protein (GFP) is a protein 
that emits green fluorescence when exposed to a radiation of ultraviolet 
wavelength range, even without the addition of substrate and cofactor. Because 
of such characteristics, the usage of GFP is widespread in both in vivo and in vitro applications. In addition, recent advances in 
biotechnology have enabled GFP to be expressed in various hosts, including 
bacteria, yeast, plants, animals, and even living-cells, for multiple purposes. 
Currently, GFP is a subject of great interest in the analytical sciences, 
especially in immunoassays for qualitative and quantitative analyses, when it 
is fused with an antibody because of the high sensitivity of GFP and 
antigen-binding specificity of antibodies. Recently the fluobody, which is a 
fusion protein of GFP with single-chain variable fragment antibody (scFv), has 
become a useful tool in various fields. We review here the applications of GFP 
as fluobodies in immunoassays.


Introduction
Green fluorescent protein (GFP) was first isolated from the jellyfish Aequorea victoria in the early 1960s [1]. The fluorescence spectra of GFP exhibit two absorption peaks at 395 nm and 475 nm (excitation), which individually emitted maximum at 508 nm and 503 nm (emission). The absorption peaks at 395 nm and 475 nm re-ble thiourea bond, FITC-labeled antibodies have been used primarily for flow cytometry and immunohistochemical staining for more than 50 years [31] [32]. This procedure, however, requires a large amount of purified protein. Moreover, the fluorophores may bind to the paratope of an antibody, which results in the antibody's partial or complete loss of reactivity. A fluobody can overcome these disadvantages because once the genes are constructed in the expression vector, the resultant protein is always expressed in a one-to-one ratio between the fluorochrome and scFv. Currently, the usage of fluobodies in vitro has expanded from the immunolabeling of cancer cells, fluorescence-activated cell sorter (FACS), and diagnosis, to fluorescent-linked immunosorbent assay (FLISA) for the qualitative/quantitative analyses [33]- [37].
The in vitro applications of GFP as a fluobody are reviewed in this article.

Immunolabeling
The fluobody possesses two characteristics: strong emission intensity and an antibody that binds to a target antigen. Immunolabeling using fluobodies is convenient method for investigating the subcellular protein and oncoprotein in living cells. When the cells are treated with the fluobody and washed, they are ready to be observed under a fluorescence microscope. Schwalbach et al. successfully expressed a fluobody against the E6 protein of human papillomavirus type 16 (E6 protein) in Escherichia coli (E. coli) and revealed that the subcellular localization and movement of E6 protein transfected in COS-7 cell lines [38]. Around the same period, Casey et al. expressed a fluobody specific to hepatitis B surface antigen (HepBsAg) in the periplasmic fraction of E. coli and revealed that the fluobody retains the functional form to recognize HepBsAg even though it is expressed as a periplasmic protein [33]. In addition, a fluobody against a series of cluster of differentiation (CD) antigen [35], p24 (human immunodeficiency virus 1) CB4-1 [39], single-chain T-cell receptor (scTCR) [40], and B-cell-activating factor of the TNF family (BAFF) [36] were successfully applied for one-step immunolabeling. More recently, a new, multi-colored newly fluobody was suggested by Markiv et al. [41]. They constructed a fluorescent antibody, where humanized scFv against p185 HER-2-ECD (Trastuzumab, Herceptin, 4D5), which is widely used in breast cancer immunotherapy [42], was fused with four kinds of monomeric red, blue, cerulean and citrine fluorescent proteins (RFP, BFP, CER, and CIT, respectively) as bridging molecules. A cell staining study using SK-BR-3 breast carcinoma cells revealed that 4D5-RFP and 4D5-CIT were found to specifically recognize p185 HER-2-ECD and effectively accumulate on the surface of the cells. Notably, a fluorescent protein other than GFP was used as a fluorescent probe. The results of this study open up the possibility of fluobodies that can be used in immunolabeling because of the various measurement wavelengths of fluorochrome.

Fluorescence-Activated Cell Sorter
Peipp et al. prepared scFv fused with two fluorescent proteins (GFP and DsRed); the fused product exhibited spectral properties that are ideal for dual-color experiments with GFP [35] [43] in which it is applied as a fluorescence-activated cell sorter (FACS). One common problem frequently encountered with a FACS is the background associated with the antigen-independent interaction between the Fc-protein and Fc receptors on various cells. These interactions result in a diminished intensity of the specific signal and subsequently cause a decrease in the signal-to-noise ratio, which complicates the collection of experimental date [44] [45]. Therefore, Peipp et al. used a fluorescent antibody because scFv antibody is composed of minimal variable regions for antigenbinding activity without Fc regions. They successfully reduced the background and enhanced the signal-tonoise ratio by a factor of 5 -10 compared with the value obtained using conventional antibodies. Their study demonstrates the possible use of fluobodies in FACS, especially when treating with cell populations expressing Fc receptors.

Fluorescence-Linked Immunosorbent Assay
FLISA can be used for both large molecules and small molecules [46] [47]. Indirect FLISA (iFLISA) is suitable for large molecules such as proteins and peptides, whereas indirect competitive FLISA (icFLISA) is appropriate for small molecules such as natural products, agrochemicals, pesticides, and herbicides. The advantages of FLISA over the conventional enzyme-linked immunosorbent assay (ELISA) are its rapidity and sensitivity. Conventional ELISA using an antibody (i.e., monoclonal antibody, polyclonal antibody, or scFv antibody) re-quires the following five steps, and the assay requires approximately 4.5 hours: 1) fixation of a coated antigen, 2) a blocking step to prevent the plate from adsorbing non-specific proteins, 3) the primary antibody reaction, 4) the secondary antibody reaction, and 5) the enzyme-substrate reaction. However, in the case of FLISA performed using a fluobody, the assay is completed within 3 hours because the time-consuming and costly secondary antibody reaction and the subsequent enzyme-substrate reaction can be avoided. In addition, sensitive FLISA can be performed by taking advantage of the strong fluorescence intensity of GFP [48]. Up to date, ic-FLISA can be developed to detect/determine small molecules, which include picloram [49], s-triazine [50], 5-methyl 2'-deoxycytidine [51] and bioactive natural product, plumbagin (PL) and ginsenoside Re (GRe) as previously described in our group [52]- [54]. The icFLISA for PL and GRe shows a lower limit of determination than conventional icELISA. In our study, we assessed the formation of a fluobody has been assessed by constructing two chimera proteins of scFv fused at the C-terminus of GFP (C-fluobody) and the N-terminus of GFP (N-fluobody). In both cases, the fluorescence intensity of the C-fluobody was superior to that of the N-fluobody. These results are supported by the findings of other groups [33] [51]. The difference in fluorescence intensity between the two can be accounted for the flexibility of the linker peptide fusing GFP and the scFv antibody. The 10 amino acids (30 bp) of the C-terminus of the GFP are well known to be flexible sequence [6] and can function as an additional linker in the C-fluobody fusing scFv at the C-terminus of GFP. Because the length of the linker peptide of fluobody was designed to contain 10 amino acids of (Gly 4 Ser) 2 encoded by 30 bp, the linker sequence of the C-fluobody is equivalent to twice as long as that of the N-fluobody. Our results showed that the flexibility of the linker strongly affects the function of GFP in the fluobody.
Recently, a new type of FLISA has been developed by Ferrara et al. [55]. In their fluobody, scFv was expressed with a tag composed of a small domain of GFP (strand 11, residues 215 -230; GFP11) with retaining its antigen-binding activity. The complementary GFP fragment (1 -10, residues 1 -214) is expressed separately. Neither fragment alone is fluorescent. Only when they are mixed, the small and large GFP fragments spontaneously associate, resulting in reconstitution of the fluorophore and fluorescence. This approach allows both the antigen and the scFv concentrations to be determined.

Conclusion
In this review, the applications of GFP as a fluobody have been described. Although the fluobody was defined as a chimera protein of GFP with scFv in this article, variations of fluobodies can be designed for different purposes. The type of fluorescent protein used can be expanded from GFP to UV, blue, cyan, yellow, orange, and red fluorescent proteins. The varieties of known fluorescent proteins have been well reviewed [56]. In addition, the antibody can be changed from scFv to fragment antigen-binding (Fab) (~55 kDa), Fab 2 (bispecific; ~110 kDa), Fab 3 (trispecific; ~165 kDa), diabody (bispecific; ~50 kDa), triabody (trivalent; ~75 kDa), tetrabody (tetravalent; ~100 kDa), bis-scFv (bispecific; ~55 kDa), and minibody (bivalent; ~75 kDa) antibodies [57]. These combinations, in conjunction with different linker designs, open up the use of fluobodies more in various fields.