Tri-part Nanoluciferase: a novel strategy for a serological assay

Protein-fragment complementation assays (PCAs) are a common approach to detect protein-protein interactions (PPIs). In PCAs, a marker protein is split into two fragments which are then respectively fused to two partner proteins. Interaction of the partner proteins brings the marker fragments into close proximity, allowing reconstitution of the marker and consequent restoration of its activity. This activity, in turn, results in production of a signal representing the interaction between the two partners under investigation.

A variety of proteins including various enzymes, transcription factors, ubiquitin, fluorescent proteins and luciferases have been used as markers in different PCA systems. Among them, NanoLuc luciferase (NLuc) was originally engineered from the deep sea shrimp Oplophorus gracilirostris [1]. It is characterized by its small size, bright luminescence and glow-type kinetics. Since it was developed, NLuc has been used in a wide range of bioluminescence applications. NanoLuc binary technology (NanoBiT) is a version of split NLuc including two fragments: LgBiT (18 kDa) and SmBiT (11 amino acids) [2]. SmBiT is derived from the C-terminal part (β10) of NLuc. NanoBiT has proven to be a powerful tool to study PPIs, however it still possesses some potential limitations. For instance, LgBiT is relatively bulky and cannot be used as a convenient short peptide tag. Additionally, the residual affinity between LgBiT and SmBiT can lead to high background signal under certain conditions.

Five years ago, Shawn Owen’s group at the University of Utah developed a tri-part split NLuc (tNLuc) strategy in which LgBiT is further split into two parts: the Δ11S fragment and the β9 peptide [3]. β9 contains 11 amino acids and forms a β sheet with neighboring residues, including β10, in the full NLuc structure. In this approach, the β9 and β10 tags are separately fused to two partner proteins. When the two partner proteins are in close proximity and Δ11S is present in the environment, β9, β10 and Δ11S reconstitute to form functional luciferase. By tagging two HER2 binders that recognize different motifs on the HER2 molecule, the system was able to detect HER2 antigen with a sensitivity at the level of 100pg/mL [3]. This work was followed by another study which similarly but independently employed the same tNLuc strategy using tags fused to VH and VL chains of an antibody to measure its corresponding antigen [4]. These two reports demonstrated the principle and feasibility of the tNLuc system, and illustrated its potential to overcome the limitations of some bipartite PCA systems. More interestingly, both studies were aimed at using tNLuc for antigen detection.

The COVID-19 pandemic represents a global health crisis of unprecedented scale. In order to help manage and combat the disease, new technologies and tools have been desperately needed. One example is high quality serological assays for detection of anti-SARS-CoV-2 antibodies. While several such assays have been developed, many have significant limitations. For example, lateral flow immunoassays suffer from low sensitivity and specificity as well as a lack of quantifiability. Automated chemiluminescence immunoassays, while capable of processing large amounts of samples, are highly dependent on expensive and specialized equipment and reagents and are therefore only suitable for centralized testing. ELISA, while possessing great performance, is not easy to carry out, usually taking 4-6 hours, requiring additional plate processing procedures and involving multiple tedious aspiration/refilling cycles. Improved serological assays would therefore be extremely useful to help better combat the pandemic.

Understanding this urgent demand, we decided to develop a novel serological assay that addressed the limitations of currently available approaches. Inspired by previous work and in collaboration with Dr. Owen, we designed a novel assay based on tNLuc, which we have called Serological Assay based on Tri-part NanoLuc (SATiN). The aim of the assay is to detect antibody isotype targeting SARS-CoV-2. We therefore used two tNLuc tagged proteins as probes: SARS-CoV-2 spike protein for idiotype specific recognition and protein G for IgG isotype recognition. Preliminary results using our assay have proven the feasibility of the design. The major obstacle to our approach was the high level of general IgG present in human serum (which also presents a challenge in ELISA) that interfered with protein G binding. Notably, however, we determined that this could be overcome by use of appropriate sample dilution, a result we confirmed both computationally and experimentally. We also carefully evaluated the assay using serum specimens from COVID-19 patients and obtained results comparable to those generated with ELISA and neutralizing antibody assays. Major highlights of our SATiN assay include:

  1. Easy operability. The reaction occurs in the liquid phase and does not involve any wash or buffer exchange step which are required in ELISA.
  2. Fast performance. The reaction takes only one hour in contrast to the typical 4-6 hours minimum required for ELISA.
  3. Cost effective. Except for a microplate luminometer, no specialized equipment is required. It is therefore suitable for both centralized and community-based testing.
  4. Good sensitivity and specificity comparable to ELISA.
  5. The readouts are correlated to neutralizing antibody assays.

We believe that SATiN can serve as a useful tool for both disease and vaccine management and make a significant contribution to combating the COVID-19 pandemic.



  1. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 7, 1848-57 (2012).
  2. Dixon AS, Schwinn MK, Hall MP, Zimmerman K, Otto P, Lubben TH, Butler BL, Binkowski BF, Machleidt T, Kirkland TA, Wood MG, Eggers CT, Encell LP, Wood KV. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol. 11, 400-8 (2016).
  3. Dixon, A. S., Kim, S. J., Baumgartner, B. K., Krippner, S. & Owen, S. C. A Tri-part Protein Complementation System Using Antibody-Small Peptide Fusions Enables Homogeneous Immunoassays. Rep. 7, 8186 (2017).
  4. Ohmuro-Matsuyama, Y. & Ueda, H. Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation. Chem. 90, 3001–3004 (2018).