Simple, Rapid Chemical Labeling and Screening of Antibodies with Luminescent Peptides

Abstract

Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.

Development of Luminescent Biosensors for Calprotectin

“…NanoBiT-based technology has recently been developed with various synthetic scaffolds and applied for different tests, including but not limited to SARS-CoV-2 (COVID-19), , Clostridium difficile infections, , and botulinum neurotoxins . While efforts have been reported in combining NanoBiT with full-length antibodies, − the most common and versatile protein-based binder so far, such antibody engineering usually relies on chemical conjugation of antibody with LgBiT or SmBiT, which necessitates not only the costly production of antibodies, but also extensive purifications due to the heterogeneity of products. While primary efforts suggested Fab binder tagged with a short peptide (peptide β9 from NanoLuc) is compatible with the NanoBiT system, we reasoned that directly fusing NanoBiT with Fab could address the challenges associated with antibody conjugation, thereby fully leveraging Fab’s versatility.…”

Development of a rapid, simple, and sensitive point-of-care technology platform utilizing ternary NanoLuc

“…HiBiT, a high-affinity strand 10 peptide, has been identified as the preferred strand 10 peptide (β10) based on luminescent signal output in the ternary system and was carried through for these experiments. We further evolved the strand 9 (β9) derived peptide as well as LgTrip to overcome poor aqueous solubility and improve protein stability ( Hall et al, 2021 ; Kincaid et al, 2022 ; Supplementary Figure 1 ). To create the chemical conjugation reporter tags, we relied on HaloTag-mediated chemical labeling of a pair of complementary antibodies that recognized non-overlapping epitopes of the target analyte ( Nath et al, 2017 ).…”

“…Using this model system, we showcased another accommodating assay feature of the ternary, split-Nluc platform, namely the ability to append β9 and β10 peptides as genetic fusions rather than the chemical labeling approach used in the antigen assay. This and a recently published study have focused on transitioning the ternary Nluc technology from HaloTag-based labeling approach to direct peptide labeling and genetic fusions to enable flexible labeling options for different targets and model systems( Kincaid et al, 2022 ). The direct labeling and genetic fusion methods use smaller peptide tags that are bioinert and can be easily appended anywhere on targets of interest.…”

“…Split versions of Nluc have been used as binary or ternary complementation reporters for the sensitive detection of analytes using several configurations ( Dixon et al, 2016 , 2017 ; Schwinn et al, 2018 ; Elledge et al, 2020 ; Hwang et al, 2020 ; Hall et al, 2021 ; Kainulainen et al, 2021 ; Ni et al, 2021 ; Yao et al, 2021 ; Kincaid et al, 2022 ). These examples highlighted the benefits of using bioluminescence for analyte detection providing sensitive assays with simplified workflows.…”

Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations

“…NanoLuc (Nluc) is an engineered bioluminescent protein developed by Promega in 2012 with numerous applications in molecular and cellular biology. − Although other bioluminescent probes are available, including widely used Firefly Luciferase (Fluc) and Renilla Luciferase (Rluc), Nluc has several important advantages, such as a smaller size, greatly increased brightness, and significantly increased structural and enzymatic stability. ,,,,,,,, Nluc originated from a small subunit of the deep sea shrimp Oplophorus luciferase (OLuc). ,,− This subunit, OLuc19, is bioluminescent, reacting in an ATP-independent reaction with coelenterazine ,,,, producing blue light, which is used by the shrimp as a self-defense mechanism against predators. Since OLuc19 was unstable and aggregated when expressed in Escherichia coli cells, various steps of amino acid mutations and substrate optimization were performed to stabilize this 19 kDa subunit.…”