Customizable OpenGUS immunoassay: a homogeneous detection system using split β-glucuronidase and label-free antibody
DOI:
https://doi.org/10.51094/jxiv.511Keywords:
immunoassay, β-glucuronidase, antibody, Cry j 1, C-creative protein, lactoferrin, homogeneousAbstract
We developed a customizable OpenGUS immunoassay for rapid and sensitive detection of analytes, eliminating the need for any antibody modifications. This assay employs a label-free whole antibody(ies), an antibody-binding domain derived from Staphylococcal protein A, and a split β-glucuronidase (GUS) mutant, allowing for the replacement of antibodies to establish an immunoassay for various targeted antigens. The working principle is that OpenGUS probe, the fusion protein of antibody-binding protein and split GUS mutant, converts the antibody-antigen interaction into GUS activation in a one-pot reaction. The split GUS mutant with decreased background activation was generated by screening several mutations at a diagonal interface residue H514. We optimized reaction buffer compositions, including organic solvent addition, salt concentrations, and surfactant concentrations, to enhance the signal/background ratio of the assay. In the optimal condition, we successfully customized OpenGUS fluorogenic immunoassays for Japanese cedar pollen allergen Cry j 1, human C-creative protein, and human lactoferrin with over 10–20-fold maximum fluorescence responses with picomolar to low nanomolar level detection limit within 15 min reaction time, by simply using commercially available IgGs. Moreover, in the absence of a fluorometer such as outdoors or at home, analytes can be detected using a simple smartphone or even the naked eye, with a pen-type UV-LED as the light source. We believe that the customizable OpenGUS immunoassay will pave new ways for the prompt development of rapid and sensitive homogeneous immunoassays for point-of-care diagnostics, high-throughput testing, and on-site environmental assessment applications.
Conflicts of Interest Disclosure
B.Z., T.Y., H.U., and T.K. received honoraria from HikariQ Health, Inc. for another unrelated project.Downloads *Displays the aggregated results up to the previous day.
References
Boguszewska, K., Szewczuk, M., Urbaniak, S. and Karwowski, B.T. (2019). Review: immunoassays in DNA damage and instability detection. Cell Mol Life Sci 76, 4689-4704.
Dai, Y.C., Sato, Y., Zhu, B., Kitaguchi, T., Kimura, H., Ghadessy, F.J. and Ueda, H. (2022). Intra Q-body: an antibody-based fluorogenic probe for intracellular proteins that allows live cell imaging and sorting. Chemical Science 13, 9739-9748.
Voller, A., Bidwell, D.E. and Bartlett, A. (1976). Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull World Health Organ 53, 55-65.
Zhu, B. et al. (2022). Rapid and sensitive SARS-CoV-2 detection using a homogeneous fluorescent immunosensor Quenchbody with crowding agents. Analyst 147, 4971-4979.
Samarajeewa, U., Wei, C.I., Huang, T.S. and Marshall, M.R. (1991). Application of immunoassay in the food industry. Crit Rev Food Sci Nutr 29, 403-434.
Su, J. et al. (2022). The Patrol Yeast: A new biosensor armed with antibody-receptor chimera detecting a range of toxic substances associated with food poisoning. Biosens Bioelectron 219, 114793.
Ahn, K.C., Kim, H.J., McCoy, M.R., Gee, S.J. and Hammock, B.D. (2011). Immunoassays and biosensors for monitoring environmental and human exposure to pyrethroid insecticides. J Agric Food Chem 59, 2792-2802.
Berson, S.A., Yalow, R.S., Bauman, A., Rothschild, M.A. and Newerly, K. (1956). Insulin-I131 metabolism in human subjects: demonstration of insulin binding globulin in the circulation of insulin treated subjects. J Clin Invest 35, 170-190.
Engvall, E. and Perlmann, P. (1972). Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J Immunol 109, 129-135.
Koczula, K.M. and Gallotta, A. (2016). Lateral flow assays. Essays Biochem 60, 111-120.
Banerjee, R. and Jaiswal, A. (2018). Recent advances in nanoparticle-based lateral flow immunoassay as a point-of-care diagnostic tool for infectious agents and diseases. Analyst 143, 1970-1996.
Takkinen, K. and Zvirbliene, A. (2019). Recent advances in homogenous immunoassays based on resonance energy transfer. Curr Opin Biotechnol 55, 16-22.
Rani, A.Q., Zhu, B., Ueda, H. and Kitaguchi, T. (2023). Recent progress in homogeneous immunosensors based on fluorescence or bioluminescence using antibody engineering. Analyst 148, 1422-1429.
Ni, Y. et al. (2021). A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays. Nat Commun 12, 4586.
Akhavan-Tafti, H. et al. (2013). A homogeneous chemiluminescent immunoassay method. J Am Chem Soc 135, 4191-4194.
Ullman, E.F., Schwarzberg, M. and Rubenstein, K.E. (1976). Fluorescent excitation transfer immunoassay. A general method for determination of antigens. J Biol Chem 251, 4172-4178.
Ueda, H., Kubota, K., Wang, Y., Tsumoto, K., Mahoney, W., Kumagai, I. and Nagamune, T. (1999). Homogeneous noncompetitive immunoassay based on the energy transfer between fluorolabeled antibody variable domains (open sandwich fluoroimmunoassay). Biotechniques 27, 738-742.
Arai, R., Nakagawa, H., Tsumoto, K., Mahoney, W., Kumagai, I., Ueda, H. and Nagamune, T. (2001). Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer. Anal Biochem 289, 77-81.
Yu, X., Wen, K., Wang, Z., Zhang, X., Li, C., Zhang, S. and Shen, J. (2016). General Bioluminescence Resonance Energy Transfer Homogeneous Immunoassay for Small Molecules Based on Quantum Dots. Anal Chem 88, 3512-3520.
Su, J., Dong, J.H., Kitaguchi, T., Ohmuro-Matsuyama, Y. and Ueda, H. (2018). Noncompetitive homogeneous immunodetection of small molecules based on beta-glucuronidase complementation (vol 143, pg 2096, 2018). Analyst 143, 3499-3499.
Su, J.L., Beh, C., Ohmuro-Matsuyama, Y., Kitaguchi, T., Hoon, S. and Ueda, H. (2019). Creation of stable and strictly regulated enzyme switch for signal-on immunodetection of various small antigens. Journal of Bioscience and Bioengineering 128, 677-682.
Zhu, B., Qian, C., Tang, H.X., Kitaguchi, T. and Ueda, H. (2023). Creating a Thermostable beta-Glucuronidase Switch for Homogeneous Immunoassay by Disruption of Conserved Salt Bridges at Diagonal Interfaces. Biochemistry 62, 309-317.
Geddie, M.L. and Matsumura, I. (2007). Antibody-induced oligomerization and activation of an engineered reporter enzyme. J Mol Biol 369, 1052-1059.
Jansson, B., Uhlen, M. and Nygren, P.A. (1998). All individual domains of staphylococcal protein A show Fab binding. Fems Immunology and Medical Microbiology 20, 69-78.
Braisted, A.C. and Wells, J.A. (1996). Minimizing a binding domain from protein A. Proc Natl Acad Sci U S A 93, 5688-5692.
Scheffel, J., Kanje, S., Borin, J. and Hober, S. (2019). Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification. MAbs 11, 1492-1501.
Ohmuro-Matsuyama, Y., Chung, C.I. and Ueda, H. (2013). Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments. BMC Biotechnol 13, 31.
Asakura, T., Adachi, K. and Schwartz, E. (1978). Stabilizing Effect of Various Organic-Solvents on Protein. Journal of Biological Chemistry 253, 6423-6425.
Arakawa, T., Kita, Y. and Timasheff, S.N. (2007). Protein precipitation and denaturation by dimethyl sulfoxide. Biophys Chem 131, 62-70.
Lindman, S., Xue, W.F., Szczepankiewicz, O., Bauer, M.C., Nilsson, H. and Linse, S. (2006). Salting the charged surface: pH and salt dependence of protein G B1 stability. Biophys J 90, 2911-2921.
Pepys, M.B. and Hirschfield, G.M. (2003). C-reactive protein: a critical update. J Clin Invest 111, 1805-1812.
Jacobs, J.F., van der Molen, R.G., Bossuyt, X. and Damoiseaux, J. (2015). Antigen excess in modern immunoassays: to anticipate on the unexpected. Autoimmun Rev 14, 160-167.
Kell, D.B., Heyden, E.L. and Pretorius, E. (2020). The Biology of Lactoferrin, an Iron-Binding Protein That Can Help Defend Against Viruses and Bacteria. Front Immunol 11, 1221.
Ponzini, E., Scotti, L., Grandori, R., Tavazzi, S. and Zambon, A. (2020). Lactoferrin Concentration in Human Tears and Ocular Diseases: A Meta-Analysis. Invest Ophthalmol Vis Sci 61, 9.
Reseco, L., Atienza, M., Fernandez-Alvarez, M., Carro, E. and Cantero, J.L. (2021). Salivary lactoferrin is associated with cortical amyloid-beta load, cortical integrity, and memory in aging. Alzheimers Research & Therapy 13, 150.
Koshi, R., Kotani, K., Ohtsu, M., Yoshinuma, N. and Sugano, N. (2018). Application of Lactoferrin and alpha1-Antitrypsin in Gingival Retention Fluid to Diagnosis of Periodontal Disease. Dis Markers 2018, 4308291.
Bagby, G.C., Jr. and Bennett, R.M. (1982). Feedback regulation of granulopoiesis: polymerization of lactoferrin abrogates its ability to inhibit CSA production. Blood 60, 108-112.
Levay, P.F. and Viljoen, M. (1995). Lactoferrin: a general review. Haematologica 80, 252-267.
Rosa, L., Cutone, A., Lepanto, M.S., Paesano, R. and Valenti, P. (2017). Lactoferrin: A Natural Glycoprotein Involved in Iron and Inflammatory Homeostasis. Int J Mol Sci 18, 1985.
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Submitted: 2023-09-25 23:17:59 UTC
Published: 2023-09-26 08:01:58 UTC
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Copyright (c) 2023
Bo Zhu
Yukihiko Yamasaki
Takanobu Yasuda
Cheng Qian
Zhirou Qiu
Hiroshi Ueda
Tetsuya Kitaguchi
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