Application of Green Fluorescent Protein in Immunoassays

DOI: 10.4236/abb.2014.56065   PDF   HTML     4,752 Downloads   6,774 Views   Citations


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.

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Sakamoto, S. , Shoyama, Y. , Tanaka, H. and Morimoto, S. (2014) Application of Green Fluorescent Protein in Immunoassays. Advances in Bioscience and Biotechnology, 5, 557-563. doi: 10.4236/abb.2014.56065.

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The authors declare no conflicts of interest.


[1] Shimomura, O., Johnson, F.H. and Saiga, Y. (1962) Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea. Journal of Cellular and Comparative Physiology, 59, 223-239.
[2] Zimmer, M. (2002) Green Fluorescent Protein (GFP): Applications, Structure, and Related Photophysical Behavior. Chemical Reviews, 102, 759-781.
[3] Niwa, H., Inouye, S., Hirano, T., Matsuno, T., Kojima, S., Kubota, M., Ohashi, M. and Tsuji, F.I. (1996) Chemical Nature of the Light Emitter of the Aequorea Green Fluorescent Protein. Proceedings of the National Academy of Sciences of the United States of America, 93, 13617-13622.
[4] Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. and Cormier, M.J. (1992) Primary Structure of the Aequorea victoria Green-Fluorescent Protein. Gene, 111, 229-233.
[5] Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. and Prasher D.C. (1994) Green Fluorescent Protein as a Marker for Gene Expression. Science, 263, 802-805.
[6] Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y. and Remington, S.J. (1996) Crystal Structure of the Aequorea victoria Green Fluorescent Protein. Science, 273, 1392-1395.
[7] Yang, F., Moss, L.G. and Phillips, G.N.J. (1996) The Molecular Structure of Green Fluorescent Protein. Nature Biotechnology, 14, 1246-1251.
[8] Sheen, J., Hwang, S.B., Niwa, Y., Kobayashi, H. and Galbraith, D.W. (1995) Green-Fluorescent Protein as a New Vital Marker in Plant Cells. Plant Journal, 8, 777-784.
[9] Cormack, B.P., Valdivia, R.H. and Falkow, S. (1996) FACS-Optimized Mutants of the Green Fluorescent Protein (GFP). Gene, 173, 33-38.
[10] Kain, S.R., Adams, M., Kondepudi, A., Yang, T.T., Ward, W.W. and Kitts, P. (1995) Green Fluorescent Protein as a Reporter of Gene Expression and Protein Localization. Biotechniques, 19, 650-655.
[11] Patterson, G.H., Knoble, S.M., Sharif, W.D., Kain, S.R. and Piston, D.W. (1997) Use of the Green Fluorescent Protein and Its Mutants in Fluorescence Microscopy. Biophysical Journal, 73, 2782-2790.
[12] Heim, R., Prasher, D.C. and Tsien, R.Y. (1994) Wavelength Mutation and Posttranslational Autoxidation of Green Fluorescent Protein. Proceedings of the National Academy of Sciences of the United States of America, 91, 12501-12504.
[13] Liu, H.S., Jan, M.S., Chou, C.K., Chen, P.H. and Ke, N.J. (1999) Is Green Fluorescent Protein Toxic to the Living Cells? Biochemical and Biophysical Research Communications, 260, 712-717.
[14] Ueda, Y., Kwok, S. and Hayashi, Y. (2013) Application of FRET Probes in the Analysis of Neuronal Plasticity. Frontiers in Neural Circuits, 7, 163.
[15] Nouar, R., Devred, F., Breuzard, G. and Peyrot, V. (2013) FRET and FRAP Imaging: Approaches to Characterise Tau and Stathmin Interactions with Microtubules in Cells. Biology of the Cell, 105, 149-161.
[16] Zeug, A., Woehler, A., Neher, E. and Ponimaskin, E.G. (2012) Quantitative Intensity-Based FRET Approaches—A Comparative Snapshot. Biophysical Journal, 103, 1821-1827.
[17] Preus, S. and Wilhelmsson, L.M. (2012) Advances in Quantitative FRET-Based Methods for Studying Nucleic Acids. ChemBioChem, 13, 1990-2001.
[18] Galbán, J., Sanz-Vicente, I., Ortega, E., del Barrio, M. and de Marcos, S. (2012) Reagentless Fluorescent Biosensors Based on Proteins for Continuous Monitoring Systems. Analytical and Bioanalytical Chemistry, 402, 3039-3054.
[19] White, J. and Stelzer, E. (1999) Photobleaching GFP Reveals Protein Dynamics Inside Live Cells. Trends in Cell Biology, 9, 61-65.
[20] Zimmer, M. (2002) Green Fluorescent Protein (GFP): Applications, Structure, and Related Photophysical Behavior. Chemical Reviews, 102, 759-781.
[21] Lippincott-Schwartz, J., Snapp, E. and Kenworthy, A. (2001) Studying Protein Dynamics in Living Cells. Nature Reviews Molecular Cell Biology, 2, 444-456.
[22] Goulbourne, C.N., Malhas, A.N., Doolittle, H., Ismail, A.T. and Vaux, D.J. (2010) Study of Molecular Dynamics Using Fluorescently Tagged Molecules in Live Cells. In: Méndez-Vilas, A. and Díaz, J., Eds., Microscopy: Science, Technology, Applications and Education, 670-678.
[23] Li, T., Bourgeois, J.P., Celli, S., Glacial, F., Le Sourd, A.M., Mecheri, S., Weksler, B., Romero, I., Couraud, P.O., Rougeon, F. and Lafaye, P. (2012) Cell-Penetrating Anti-GFAP VHH and Corresponding Fluorescent Fusion Protein VHH-GFP Spontaneously Cross the Blood-Brain Barrier and Specifically Recognize Astrocytes: Application to Brain Imaging. The FASEB Journal, 26, 3969-3979.
[24] Urakami, E., Yamaguchi, I., Asami, T., Conrad, U. and Suzuki, Y. (2008) Immunomodulation of Gibberellin Biosynthesis Using an Anti-Precursor Gibberellin Antibody Confers Gibberellin-Deficient Phenotypes. Planta, 228, 863-873.
[25] Yamaguchi, S. (2008) Gibberellin Metabolism and Its Regulation. Annual Review of Plant Biology, 59, 225-251.
[26] Ahmad, Z.A., Yeap, S.K., Ali, A.M., Ho, W.Y., Alitheen, N.B. and Hamid, M. (2012) scFv Antibody: Principles and Clinical Application. Clinical & Developmental Immunology, 2012, 980250.
[27] Beckman, R.A., Weiner, L.M. and Davis, H.M. (2007) Antibody Constructs in Cancer Therapy: Protein Engineering Strategies to Improve Exposure in Solid Tumors. Cancer, 109, 170-179.
[28] Leath 3rd, C.A., Douglas, J.T., Curiel, D.T. and Alvarez, R.D. (2004) Single-Chain Antibodies: A Therapeutic Modality for Cancer Gene Therapy (Review). International Journal of Oncology, 24, 765-771.
[29] Huang, J., Li, Y., Guo, F., Tong, Y., Wang, J., Hu, J. and Li, G. (2011) Expression of scFv SA3 against Hepatoma Fused with Enhanced Green Fluorescent Protein and Its Targeted Ability in Vivo. Zhong Nan Da Xue Xue Bao Yi Xue Ban/Journal of Central South University, 36, 979-986.
[30] Hermanson, G.T. (1996) Bioconjugate Techniques. Academic Press, San Diego, 297-364.
[31] Goldstein, G., Slizys, I.S. and Chase, M.W. (1961) Studies on Fluorescent Antibody Staining. I. Non-Specific Fluorescence with Fluorescein-Coupled Sheep Anti-Rabbit Globulins. The Journal of Experimental Medicine, 114, 89-110.
[32] Sommerville, R.G. (1967) The Production of Fluorescent Antibody Reagents for Virus Diagnosis in the Albino Mouse. Archiv für die gesamte Virusforschung, 20, 452-458.
[33] Casey, J.L., Coley, A.M., Tilley, L.M. and Foley, M. (2000) Green Fluorescent Antibodies: Novel in Vitro Tools. Protein Engineering, 13, 445-452.
[34] Souriu, C. and Hudson, P.J. (2001) Recombinant Antibodies for Cancer Diagnosis and Therapy. Expert Opinion on Biological Therapy, 1, 845-855.
[35] Peipp, M., Saul, D., Barbin, K., Bruenke, J., Zunino, S.J., Niederweis, M. and Fey, G.H. (2004) Efficient Eukaryotic Expression of Fluorescent scFv Fusion Proteins Directed against CD Antigens for FACS Applications. Journal of Immunological Methods, 285, 265-280.
[36] Cao, M., Cao, P., Yan, H.J., Ren, F., Lu, W.G., Hu, Y.L. and Zhang, S.Q. (2008) Construction and Characterization of an Enhanced GFP-Tagged Anti-BAFF scFv Antibody. Applied Microbiology and Biotechnology, 79, 423-431.
[37] Olichon, A. and Surrey, T. (2007) Selection of Genetically Encoded Fluorescent Single Domain Antibodies Engineered for Efficient Expression in Escherichia coli. The Journal of Biological Chemistry, 282, 36314-36320.
[38] Schwalbach, G., Sibler, A.P., Choulier, L., Deryckère, F. and Weiss, E. (2000) Production of Fluorescent Single-Chain Antibody Fragments in Escherichia coli. Protein Expression and Purifications, 18, 121-132.
[39] Naumann, J.M., Küttner, G. and Bureik, M. (2011) Expression and Secretion of a CB4-1 scFv-GFP Fusion Protein by Fission Yeast. Applied Biochemistry and Biotechnology, 163, 80-89.
[40] Huang, D. and Shusta, E.V. (2006) A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions. Applied and Environmental Microbiology, 72, 7748-7759.
[41] Markiv, A., Beatson, R., Burchell, J., Durvasula, R.V. and Kang, A.S. (2011) Expression of Recombinant Multi-Coloured Fluorescent Antibodies in gor¯/trxB¯ E. coli Cytoplasm. BMC Biotechnology, 11, 117.
[42] Phillips, K.A., Marshall, D.A., Haas, J.S., Elkin, E.B., Liang, S.Y., Hassett, M.J., Ferrusi, I., Brock, J.E. and Van Bebber, S.L. (2009) Clinical Practice Patterns and Cost Effectiveness of Human Epidermal Growth Receptor 2 Testing Strategies in Breast Cancer Patients. Cancer, 115, 5166-5174.
[43] Bevis, B.J. and Glick, B.S. (2002) Rapidly Maturing Variants of the Discosoma Red Fluorescent Protein (DsRed). Nature Biotechnology, 20, 83-87.
[44] Gadd, S.J. and Ashman, L.K. (1983) Binding of Mouse Monoclonal Antibodies to Human Leukaemic Cells via the Fc Receptor: A Possible Source of “False Positive” Reactions in Specificity Screening. Clinical and Experimental Immunology, 54, 811-818.
[45] Imamura, N., Ota, H. and Kuramoto, A. (1991) CD7 False-Positive Acute Myelogenous Leukemia and Promyelocytic Leukemia Cell Line HL-60: Characterization of CD7 Epitopes by Four Monoclonal Antibodies. American Journal of Hematology, 38, 72-74.
[46] Magnusson, K.E., Bartonek, E., Nordkvist, E., Sundqvist, T. and Asbrink, E. (1987) Fluorescence-Linked Immunosorbent Assay (FLISA) for Quantification of Antibodies to Food Antigens. Immunological Investigations, 16, 227-240.
[47] Velappan, N., Clements, J., Kiss, C., Valero-Aracama, R., Pavlik, P. and Bradbury, A.R. (2008) Fluorescence Linked Immunosorbant Assays Using Microtiter Plates. Journal of Immunological Methods, 336, 135-141.
[48] Sakamoto, S., Pongkitwitoon, B., Nakahara, H., Shibata, O., Shoyama, Y., Tanaka, H. and Morimoto, S. (2012) Fluobodies against Bioactive Natural Products and Their Application in Fluorescence-Linked Immunosorbent Assay. Antibodies, 1, 239-258.
[49] Kim, I.S., Shim, J.H., Suh, Y.T., Yau, K.Y.F., Hall, J.C., Trevors, J.T. and Lee, H. (2002) Green Fluorescent Protein-Labeled Recombinant Antibody for Detecting the Picloram Herbicide. Bioscience, Biotechnology, and Biochemistry, 66, 1148-1151.
[50] Oelschlaeger, P., Srikant-Iyer, S., Lange, S., Schmitt, J. and Schmid, R.D. (2002) Fluorophor-Linked Immunosorbent Assay: A Time- and Cost-Saving Method for the Characterization of Antibody Fragments Using a Fusion Protein of a Single-Chain Antibody Fragment and Enhanced Green Fluorescent Protein. Analytical Biochemistry, 309, 27-34.
[51] Ohshima, M., Inoue, K., Hayashi, H., Tsuji, D., Mizugaki, M. and Itoh, K. (2010) Generation of AcGFP Fusion with Single-Chain Fv Selected from a Phage Display Library Constructed from Mice Hyperimmunized against 5-Methyl 2’-Deoxycytidine. Protein Engineering, Design & Selection, 23, 881-888.
[52] Sakamoto, S., Taura, F., Pongkitwitoon, B., Putalun, W., Tsuchihashi, R., Kinjo, J., Tanaka, H. and Morimoto, S. (2010) Development of Sensitivity-Improved Fluorescence-Linked Immunosorbent Assay Using a Fluorescent Single-Domain Antibody against the Bioactive Naphthoquinone, Plumbagin. Analytical and Bioanalytical Chemistry, 396, 2955-2963.
[53] Sakamoto, S., Tanizaki, Y., Pongkitwitoon, B., Tanaka, H. and Morimoto, S. (2011) A Chimera of Green Fluorescent Protein with Single Chain Variable Fragment Antibody against Ginsenosides for Fluorescence-Linked Immunosorbent Assay. Protein Expression and Purification, 77, 124-130.
[54] Sakamoto, S., Pongkitwitoon, B., Sasaki-Tabata, K., Putalun, W., Maenaka, K., Tanaka, H. and Morimoto, S. (2011) A Fluorescent Single Domain Antibody against Plumbagin Expressed in Silkworm Larvae for Fluorescence-Linked Immunosorbent Assay (FLISA). Analyst, 136, 2056-2063.
[55] Ferrara, F., Listwan, P., Waldo, G.S. and Bradbury, A.R. (2011) Fluorescent Labeling of Antibody Fragments Using Split GFP. PLoS One, 6, e25727.
[56] Chudakov, D.M., Matz, M.V., Lukyanov, S. and Lukyanov, K.A. (2010) Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiological Reviews, 90, 1103-1163.
[57] Holliger, P. and Hudson, P.J. (2005) Engineered Antibody Fragments and the Rise of Single Domains. Nature Biotechnology, 23, 1126-1136.

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