An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb), Fab antibody, and single-chain variable fragment (scFv) antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP) with a scFv antibody, named fluobody, was proposed as a probe for an alternative immunosorbent assay; i.e., fluorescence-linked immunosorbent assay (FLISA). In this FLISA, an even more sensitive, simple, and rapid immunoassay can be performed by detecting the highly sensitive fluorophore of GFP that is genetically and directly fused to the scFv antibody. In addition, the time- and cost-consuming secondary antibody reaction and the following enzyme-substrate reaction, necessary for conventional ELISA, can be avoided, making it possible to complete the assay more rapidly. Focusing on naturally occurring bioactive products, fluobody recognizing 1,4-naphthoquinone, plumbagin and triterpenoid saponin, ginsenosides were successfully expressed in Escherichia coli (E. coli) and applied to FLISA. The construction, the expression, and the potential use of fluobody in quantitative/qualitative analysis of bioactive natural products are reviewed in this article.
References
[1]
Yalow, R.S.; Berson, S.A. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 1960, 39, 1157–1175, doi:10.1172/JCI104130.
[2]
Lê, H.Q.A.; Sauriat-Dorizon, H.; Korri-Youssoufi, H. Investigation of SPR and electrochemical detection of antigen with polypyrrole functionalized by biotinylated single-chain antibody: A review. Anal. Chim. Acta 2010, 674, 1–8, doi:10.1016/j.aca.2010.06.008.
[3]
Cullen, D.C.; Brown, R.G.W.; Lowe, C.R. Detection of immuno-complex formation via surface plasmon resonance on gold-coated diffraction gratings. Biosensors 1987, 3, 211–225, doi:10.1016/0265-928X(87)85002-2.
[4]
Dangl, J.L.; Herzenberg, L.A. Selection of hybridomas and hybridoma variants using the fluorescence activated cell sorter. J. Immunol. Methods 1982, 52, 1–14, doi:10.1016/0022-1759(82)90344-1.
[5]
Lanier, L.L.; Warner, N.L. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. J. Immunol. Methods 1981, 47, 25–30, doi:10.1016/0022-1759(81)90253-2.
[6]
Putalun, W.; Fukuda, N.; Tanaka, H.; Shoyama, Y. A one-step immunochromatographic assay for detecting ginsenosides Rb1 and Rg1. Anal. Bioanal. Chem. 2004, 378, 1338–1341, doi:10.1007/s00216-003-2408-x.
[7]
Putalun, W.; Morinaga, O.; Tanaka, H.; Shoyama, Y. Development of a one-step immunochromatographic strip test for the detection of sennosides A and B. Phytochem. Anal. 2004, 15, 112–116, doi:10.1002/pca.752.
[8]
Carnegie, P.R.; Pacheco, G. Immunochromatography: A combination of chromatography and immunodiffusion on a micro-scale. Proc. Soc. Exp. Biol. Med. 1964, 117, 137–141.
[9]
Shan, S.; Tanaka, H.; Shoyama, Y. Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid. Anal. Chem. 2001, 73, 5784–5790, doi:10.1021/ac0106997.
[10]
Morinaga, O.; Zhu, S.; Tanaka, H.; Shoyama, Y. Visual detection of saikosaponins by on-membrane immunoassay and estimation of traditional Chinese medicines containing Bupleuri radix. Biochem. Biophys. Res. Commun. 2006, 346, 687–692, doi:10.1016/j.bbrc.2006.05.129.
[11]
Tanaka, H.; Fukuda, N.; Shoyama, Y. Eastern blotting and immunoaffinity concentration using monoclonal antibody for ginseng saponins in the field of traditional Chinese medicines. J. Agric. Food Chem. 2007, 55, 3783–3787, doi:10.1021/jf063457m.
[12]
Shoyama, Y. Pharmacognosical study during 40 years. Yakugaku Zasshi 2007, 127, 1593–1620, doi:10.1248/yakushi.127.1593.
[13]
K？hler, G.; Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975, 256, 495–497, doi:10.1038/256495a0.
Karawajew, L.; Micheel, B.; Behrsing, O.; Gaestel, M. Bispecific antibody-producing hybrid hybridomas selected by a fluorescence activated cell sorter. J. Immunol. Methods 1987, 96, 265–270, doi:10.1016/0022-1759(87)90323-1.
[16]
Sakamoto, S.; Putalun, W.; Tsuchihashi, R.; Morimoto, S.; Kinjo, J.; Tanaka, H. Development of an enzyme-linked immunosorbent assay (ELISA) using highly-specific monoclonal antibodies against plumbagin. Anal. Chim. Acta 2008, 607, 100–105, doi:10.1016/j.aca.2007.11.021.
[17]
Morinaga, O.; Tanaka, H.; Shoyama, Y. Enzyme-linked immunosorbent assay for the determination of total ginsenosides in ginseng. Anal. Lett. 2006, 39, 287–296, doi:10.1080/00032710500476979.
[18]
Nguyen, A.T.; Malonne, H.; Duez, P.; Vanhaelen-Fastre, R.; Vanhaelen, M.; Fontaine, J. Cytotoxic constituents from Plumbago zeylanica. Fitoterapia 2004, 75, 500–504, doi:10.1016/j.fitote.2004.03.009.
[19]
Gangopadhyay, M.; Dewanjee, S.; Bhattacharyya, S.; Bhattacharya, S. Effect of different strains of Agrobacterium rhizogenes and nature of explants on Plumbago indica hairy root culture with special emphasis on root biomass and plumbagin production. Nat. Prod. Commun. 2010, 5, 1913–1916.
[20]
Serrilli, A.M.; Sanfilippo, V.; Ballero, M.; Sanna, C.; Poli, F.; Scartezzini, P.; Serafini, M.; Bianco, A. Polar and antioxidant fraction of Plumbago europaea L., a spontaneous plant of Sardinia. Nat. Prod. Res. 2010, 24, 633–639, doi:10.1080/14786410902941329.
[21]
Sandur, S.K.; Ichikawa, H.; Sethi, G.; Ahn, K.S.; Aggarwal, B.B. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-κB activation and NF-κB-regulated gene products through modulation of p65 and IκBα kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents. J. Biol. Chem. 2006, 281, 17023–17033, doi:10.1074/jbc.M601595200.
[22]
Itogawa, M.; Takeya, K.; Furukawa, H. Cardiotonic action of plumbagin on guinea-pig papillary muscle. Planta Med. 1991, 57, 317–319, doi:10.1055/s-2006-960106.
[23]
Paiva, de S.R.; Figueiredo, M.R.; Arag？o, T.V.; Kaplan, M.A.C. Antimicrobial activity in vitro of plumbagin isolated from Plumbago species. Mem. Inst. Oswaldo. Cruz. 2003, 98, 959–961, doi:10.1590/S0074-02762003000700017.
[24]
Bhargava, S.K. Effects of plumbagin on reproductive function of male dog. Indian J. Exp. Biol. 1984, 22, 153–156.
[25]
Srinivasan, L.; Mathew, N.; Muthuswamy, K. In vitro antifilarial activity of glutathione S-transferase inhibitors. Parasitol. Res. 2009, 105, 1179–1182, doi:10.1007/s00436-009-1534-6.
Stensen, W.; Jensen, E. High-performance liquid chromatrographic separations of naphthoquinones and their derivatives. J. Chromatogr. A 1994, 659, 87–93, doi:10.1016/0021-9673(94)85009-7.
[28]
Babula, P.; Mikelova, R.; Adam, V.; Kizek, R.; Havel, L.; Sladky, Z. Using of liquid chromatography coupled with diode array detector for determination of naphthoquinones in plants and for investigation of influence of pH of cultivation medium on content of plumbagin in Dionaea muscipula. J. Chromatogr. B 2006, 842, 28–35, doi:10.1016/j.jchromb.2006.05.009.
[29]
Hsieh, Y.J.; Lin, L.C.; Tsai, T.H. Determination and identification of plumbagin from the roots of Plumbago zeylanica L. by liquid chromatography with tandem mass spectrometry. J. Chromatogr. A 2005, 1083, 141–145, doi:10.1016/j.chroma.2005.06.030.
Susin, S.A.; Lorenzo, H.K.; Zamzami, N.; Marzo, I.; Brenner, C.; Larochette, N.; Prévost, M.; Alzari, P.M.; Kroemer, G. Mitochondrial release of caspase-2 and -9 during the apoptotic process. J. Exp. Med. 1999, 189, 381–394, doi:10.1084/jem.189.2.381.
[33]
Luo, J.Z.; Luo, L. Ginseng on hyperglycemia: Effects and mechanisms. Evid. Based Complement Altern. Med. 2009, 6, 423–427, doi:10.1093/ecam/nem178.
[34]
Xie, J.T.; Mchendale, S.; Yuan, C.S. Ginseng and diabetes. Am. J. Chin. Med. 2005, 33, 397–404, doi:10.1142/S0192415X05003004.
[35]
Liu, L.; Huang, J.; Hu, X.; Li, K.; Sun, C. Simultaneous determination of ginsenoside (G-Re, G-Rg1, G-Rg2, G-F1, G-Rh1) and protopanaxatriol in human plasma and urine by LC-MS/MS and its application in a pharmacokinetics study of G-Re in volunteers. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2011, 879, 2011–2017, doi:10.1016/j.jchromb.2011.05.018.
[36]
Li, L.; Luo, G.A.; Liang, Q.L.; Hu, P.; Wang, Y.M. Rapid qualitative and quantitative analyses of Asian ginseng in adulterated American ginseng preparations by UPLC/Q-TOF-MS. J. Pharm. Biomed. Anal. 2010, 52, 66–72, doi:10.1016/j.jpba.2009.12.017.
[37]
Kanazawa, H.; Nagata, Y.; Matsushima, Y.; Tomoda, M.; Takai, N. Simultaneous determination of ginsenosides and saikosaponins by high-performance liquid chromatography. J. Chromatogr. 1990, 507, 327–332, doi:10.1016/S0021-9673(01)84210-2.
[38]
Fukuda, N.; Tanaka, H.; Shoyama, Y. Applications of ELISA, western blotting and immunoaffinity concentration for survey of ginsenosides in crude drugs of Panax species and traditional Chinese herbal medicines. Analyst 2000, 125, 1425–1429, doi:10.1039/b002932g.
[39]
Deng, G.F.; Wang, D.L.; Meng, M.X.; Hu, F.; Yao, T.W. Simultaneous determination of notoginsenoside R1, ginsenoside Rg1, Re, Rb1 and icariin in rat plasma by ultra-performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009, 877, 2113–2122, doi:10.1016/j.jchromb.2009.06.003.
[40]
Sakamoto, S.; Taura, F.; Putalun, W.; Pongkitwitoon, B.; Tsuchihashi, R.; Morimoto, S.; Kinjo, J.; Shoyama, Y.; Tanaka, H. Construction and expression of specificity-improved single-chain variable fragments against the bioactive naphthoquinone, plumbagin. Biol. Pharm. Bull. 2009, 32, 434–439, doi:10.1248/bpb.32.434.
[41]
Pongkitwitoon, B.; Sakamoto, S.; Morinaga, O.; Juengwatanatrakul, T.; Shoyama, Y.; Tanaka, H.; Morimoto, S. Single-chain variable fragment antibody against ginsenoside Re as an effective tool for the determination of ginsenosides in various ginsengs. J. Nat. Med. 2011, 65, 24–30, doi:10.1007/s11418-010-0446-1.
[42]
Sakamoto, S.; Taura, F.; Tsuchihashi, R.; Putalun, W.; Kinjo, J.; Tanaka, H.; Morimoto, S. Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in Sf9 insect cell. Hybridoma (Larchmt) 2010, 29, 481–488, doi:10.1089/hyb.2010.0052.
[43]
Sakamoto, S.; Pongkitwitoon, B.; Nakamura, S.; Maenaka, K.; Tanaka, H.; Morimoto, S. Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides. J. Biochem. 2010, 148, 335–340, doi:10.1093/jb/mvq072.
[44]
Hermanson, G.T. Bioconjugate Techniques; Academic Press: San Diego, CA, USA, 1996; pp. 297–364.
[45]
Goldstein, G.; Slizys, I.S.; Chase, M.W. Studies on fluorescent antibody staining. I. Non-specific fluorescence with fluorescein-coupled sheep anti-rabbit globulins. J. Exp. Med. 1961, 114, 89–110, doi:10.1084/jem.114.1.89.
[46]
Sommerville, R.G. The production of fluorescent antibody reagents for virus diagnosis in the albino mouse. II. Coupling of mouse immune globulin with fluorescein isothiocyanate (FITC). Arch. Gesamte. Virusforsch. 1967, 20, 452–458, doi:10.1007/BF01275226.
[47]
Souriu, C.; Hudson, P.J. Recombinant antibodies for cancer diagnosis and therapy. Expert Opin. Biol. Ther. 2001, 1, 845–855, doi:10.1517/14712598.1.5.845.
[48]
Casey, J.L.; Coley, A.M.; Tilley, L.M.; Foley, M. Green fluorescent antibodies: novel in vitro tools. Protein Eng. 2000, 13, 445–452, doi:10.1093/protein/13.6.445.
[49]
Peipp, M.; Saul, D.; Barbin, K.; Bruenke, J.; Zunino, S.J.; Niederweis, M.; Fey, G.H. Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications. J. Immunol. Methods 2004, 285, 265–280, doi:10.1016/j.jim.2003.12.001.
[50]
Cao, M.; Cao, P.; Yan, H.J.; Ren, F.; Lu, W.G.; Hu, Y.L.; Zhang, S.Q. Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody. Appl. Microbiol. Biotechnol. 2008, 79, 423–431, doi:10.1007/s00253-008-1447-9.
[51]
Olichon, A.; Surrey, T. Selection of genetically encoded fluorescent single domain antibodies engineered for efficient expression in Escherichia coli. J. Biol. Chem. 2007, 282, 36314–36320, doi:10.1074/jbc.M704908200.
[52]
Kim, I.S.; Shim, J.H.; Suh, Y.T.; Yau, K.Y.F.; Hall, J.C.; Trevors, J.T.; Lee, H. Green fluorescent protein-labeled recombinant antibody for detecting the picloram herbicide. Biosci. Biotechnol. Biochem. 2002, 66, 1148–1151, doi:10.1271/bbb.66.1148.
[53]
Oelschlaeger, P.; Srikant-Iyer, S.; Lange, S.; Schmitt, J.; Schmid, R.D. 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. Anal. Biochem. 2002, 309, 27–34.
[54]
Sakamoto, S.; Taura, F.; Pongkitwitoon, B.; Putalun, W.; Tsuchihashi, R.; Kinjo, J.; Tanaka, H.; Morimoto, S. Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin. Anal. Bioanal. Chem. 2010, 396, 2955–2963, doi:10.1007/s00216-010-3535-9.
[55]
Sakamoto, S.; Tanizaki, Y.; Pongkitwitoon, B.; Tanaka, H.; Morimoto, S. A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay. Protein Expr. Purif. 2011, 77, 124–130, doi:10.1016/j.pep.2011.01.010.
[56]
Sakamoto, S.; Pongkitwitoon, B.; Sasaki-Tabata, K.; Putalun, W.; Maenaka, K.; Tanaka, H.; Morimoto, S. A fluorescent single domain antibody against plumbagin expressed in silkworm larvae for fluorescence-linked immunosorbent assay (FLISA). Analyst 2011, 136, 2056–2063.
[57]
Horton, R.M.; Hunt, H.D.; Ho, S.N.; Pullen, J.K.; Pease, L.R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 1989, 77, 61–68, doi:10.1016/0378-1119(89)90359-4.
[58]
Umetsu, M.; Tsumoto, K.; Hara, M.; Ashish, K.; Goda, S.; Adschiri, T.; Kumagai, I. How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system. Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment. J. Biol. Chem. 2003, 278, 8979–8987.
[59]
Ohshima, M.; Inoue, K.; Hayashi, H.; Tsuji, D.; Mizugaki, M.; Itoh, K. Generation of AcGFP fusion with single-chain Fv selected from a phage display library constructed from mice hyperimmunized against 5-methyl 2'-deoxycytidine. Protein Eng. Des. Sel. 2010, 23, 881–888, doi:10.1093/protein/gzq066.
[60]
Ormo, M.; Cubitt, A.B.; Kallio, K.; Gross, L.A.; Tsien, R.Y.; Remington, S.J. Crystal structure of Aequorea victoria green fluorescent protein. Science 1996, 273, 1392–1395.
[61]
Friguet, B.; Chaffotte, A.F.; Djavadi-Ohaniance, L.; Goldberg, M.E. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Methods 1985, 77, 305–319, doi:10.1016/0022-1759(85)90044-4.
[62]
Ishiyama, M.; Shoyama, Y.; Murakami, H.; Shinohara, H. Production of monoclonal antibodies and development of an ELISA for solamargine. Cytotechnology 1996, 18, 153–158.
[63]
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254, doi:10.1016/0003-2697(76)90527-3.
[64]
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685, doi:10.1038/227680a0.
[65]
Weiler, E.; Zenk, M.H. Radioimmunoassay for determination of digoxin and related compounds in Digitalis lanata. Phytochemistry 1976, 15, 1537–1545, doi:10.1016/S0031-9422(00)88933-5.
[66]
Orlandi, R.; Gussow, D.H.; Jones, P.T.; Winter, G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 1989, 86, 3833–3837, doi:10.1073/pnas.86.10.3833.
