Presentation Information
[P03-385]Rapid fabrication of homogeneous bioluminescent immunosensors using Swtichbody technology and coiled-coil peptide pair
○Takanobu Yasuda1, Shun Kagamihara1, Masahiko Taguchi2, Bo Zhu1, Eriko Nango2, Tetsuya Kitaguchi1 (1. Institute of Science Tokyo (Japan), 2. Tohoku University (Japan))
Keywords:
enzyme switch,homogeneous immunoassay,antibody,luciferase,coiled-coil peptide
Immunoassays play an important role in a wide range of fields for detecting trace amounts of analytes in complex samples. To develop a simple immunoassay, we have engineered an immunosensor bioluminescent "Switchbody" which can detect antigen homogeneously.1) This sensor is fabricated by fusing a NanoLuc-derived fragment, HiBiT, to the N-terminus of an antibody. The working principle involves HiBiT being trapped within the antibody and subsequently released upon antigen binding. This release enables HiBiT to access the complementary NanoLuc fragment, LgBiT, triggering enzyme reconstitution and an increase in enzymatic activity.
However, antibodies that function as the bioluminescent Switchbody have been limited to the anti-osteocalcin antibody, KTM219. The optimal position of HiBiT that enables its trap-and-release before and after antigen binding is expected to differ among antibodies. In this study, we developed a high-throughput method utilizing the coiled-coil peptide pair E4/K4 to optimize HiBiT position, aiming to efficiently obtain Switchbodies.
To fabricate Switchbodies using E4/K4 (coiled Switchbody), we employed a KTM219 as a model antibody. E4 was fused to the N-terminus of the heavy or light chain of the antibody fragment (E4H-Fab and E4L-Fab). In parallel, two types of HiBiT-conjugated K4 peptides, K4-linker-HiBiT and HiBiT-linker-K4, were prepared with three different repeating SGGG linker lengths (0-2 repeats; L0–L2). These six peptides were mixed with E4-Fabs to assemble coiled Switchbodies. Upon addition of antigen in the presence of LgBiT and substrate, six combinations of E4-Fab/HiBiT-linker-K4 showed a 2–3-fold increase in luminescence, demonstrating that bioluminescent coiled Switchbody can be easily and rapidly fabricated by using E4/K4.
We next extend this approach to anti-thyroxine antibody, D11, which had failed to function as bioluminescent Switchbody. Among twelve total combinations of E4-Fab/peptide, E4L-Fab/K4-L1-HiBiT showed thyroxine-dependent luminescence increase, suggesting that systematically exploring a range of combinations using this approach is an effective strategy for obtaining functional bioluminescent coiled Switchbodies.
To investigate whether the coiled Switchbody can be fabricated from antibodies that recognize large molecules, we employed an anti-TNFα antibody, adalimumab. Unexpectedly, eleven out of twelve combinations showed luminescence decrease upon antigen addition, but this decrease was sufficient for quantifying antigen. We hypothesized that steric hindrance between HiBiT and TNFα restricts the access of LgBiT to HiBiT, leading to the luminescence decrease.
In conclusion, we demonstrated an efficient approach for fabricating bioluminescent Switchbodies for both small and large molecule targets. This approach lays the groundwork for the rapid and flexible development of homogeneous immunosensors applicable to a wide range of analytes.
Reference
1) T. Yasuda et al, Adv. Sci., 12, e08422 (2025)
However, antibodies that function as the bioluminescent Switchbody have been limited to the anti-osteocalcin antibody, KTM219. The optimal position of HiBiT that enables its trap-and-release before and after antigen binding is expected to differ among antibodies. In this study, we developed a high-throughput method utilizing the coiled-coil peptide pair E4/K4 to optimize HiBiT position, aiming to efficiently obtain Switchbodies.
To fabricate Switchbodies using E4/K4 (coiled Switchbody), we employed a KTM219 as a model antibody. E4 was fused to the N-terminus of the heavy or light chain of the antibody fragment (E4H-Fab and E4L-Fab). In parallel, two types of HiBiT-conjugated K4 peptides, K4-linker-HiBiT and HiBiT-linker-K4, were prepared with three different repeating SGGG linker lengths (0-2 repeats; L0–L2). These six peptides were mixed with E4-Fabs to assemble coiled Switchbodies. Upon addition of antigen in the presence of LgBiT and substrate, six combinations of E4-Fab/HiBiT-linker-K4 showed a 2–3-fold increase in luminescence, demonstrating that bioluminescent coiled Switchbody can be easily and rapidly fabricated by using E4/K4.
We next extend this approach to anti-thyroxine antibody, D11, which had failed to function as bioluminescent Switchbody. Among twelve total combinations of E4-Fab/peptide, E4L-Fab/K4-L1-HiBiT showed thyroxine-dependent luminescence increase, suggesting that systematically exploring a range of combinations using this approach is an effective strategy for obtaining functional bioluminescent coiled Switchbodies.
To investigate whether the coiled Switchbody can be fabricated from antibodies that recognize large molecules, we employed an anti-TNFα antibody, adalimumab. Unexpectedly, eleven out of twelve combinations showed luminescence decrease upon antigen addition, but this decrease was sufficient for quantifying antigen. We hypothesized that steric hindrance between HiBiT and TNFα restricts the access of LgBiT to HiBiT, leading to the luminescence decrease.
In conclusion, we demonstrated an efficient approach for fabricating bioluminescent Switchbodies for both small and large molecule targets. This approach lays the groundwork for the rapid and flexible development of homogeneous immunosensors applicable to a wide range of analytes.
Reference
1) T. Yasuda et al, Adv. Sci., 12, e08422 (2025)
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