Customizable OpenGUS immunoassay: a homogeneous detection system using split β-glucuronidase and label-free antibody

Zhu, Bo; Yamasaki, Yukihiko; Yasuda, Takanobu; Qian, Cheng; Qiu, Zhirou; Ueda, Hiroshi; Kitaguchi, Tetsuya

doi:10.51094/jxiv.511

##article.authors##

Zhu, Bo Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology
Yamasaki, Yukihiko BioDynamics Laboratory Inc.
Yasuda, Takanobu Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology
Qian, Cheng Graduate School of Life Science and Technology, Tokyo Institute of Technology
Qiu, Zhirou Graduate School of Life Science and Technology, Tokyo Institute of Technology
Ueda, Hiroshi Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology
Kitaguchi, Tetsuya Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology

DOI:

https://doi.org/10.51094/jxiv.511

キーワード:

immunoassay、 β-glucuronidase、 antibody、 Cry j 1、 C-creative protein、 lactoferrin、 homogeneous

抄録

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.

利益相反に関する開示

B.Z., T.Y., H.U., and T.K. received honoraria from HikariQ Health, Inc. for another unrelated project.

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