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

Point-of-care tests are highly valuable in providing fast results for medical decisions for greater flexibility in patient care. Many diagnostic tests, such as ELISAs, that are commonly used within clinical laboratory settings require trained technicians, laborious workflows, and complex instrumentation hindering their translation into point-of-care applications. Herein, we demonstrate the use of a homogeneous, bioluminescent-based, split reporter platform that enables a simple, sensitive, and rapid method for analyte detection in clinical samples. We developed this point-of-care application using an optimized ternary, split-NanoLuc luciferase reporter system that consists of two small reporter peptides added as appendages to analyte-specific affinity reagents. A bright, stable bioluminescent signal is generated as the affinity reagents bind to the analyte, allowing for proximity-induced complementation between the two reporter peptides and the polypeptide protein, in addition to the furimazine substrate. Through lyophilization of the stabilized reporter system with the formulated substrate, we demonstrate a shelf-stable, all-in-one, add-and-read analyte-detection system for use in complex sample matrices at the point-of-care. We highlight the modularity of this platform using two distinct SARS-CoV-2 model systems: SARS-CoV-2 N-antigen detection for active infections and anti-SARS-CoV-2 antibodies for immunity status detection using chemically conjugated or genetically fused affinity reagents, respectively. This technology provides a simple and standardized method to develop rapid, robust, and sensitive analyte-detection assays with flexible assay formatting making this an ideal platform for research, clinical laboratory, as well as point-of-care applications utilizing a simple handheld luminometer.


Purification of HaloTag® Fusions to Peptide 840 and VS-HiBiT.
Colonies of KRX E. coli cells (Promega) transformed with HT-β9 (peptide 840) or HT-β10 (Vs-HiBiT) plasmids were inoculated into starter cultures containing LB broth + antibiotic and grown for 18 h at 37 °C. After, 1:100 dilutions of the overnight cultures were made with 500 mL of LB + 0.1% Rhamnose + 0.15% Glucose + antibiotic and grown for 20 h at 25 °C. The overexpressed cells were pelleted and re-suspended in a lysis buffer containing Phosphate-buffered saline (PBS) with 0.2 mg/mL Lysozyme (Sigma) and 1X Protease Inhibitor Cocktail (Promega). A lysate was prepared by subjecting the sample to three freeze/thaw cycles (between -80°C and room temperature). After the final thaw, 0.001 U/mL RNase-free RQ1 DNase (Promega) was added to the sample and incubated on ice for 30 min. The lysate was centrifuged, and the supernatant was collected and HaloTag-VS-HiBiT was purified using batch methodology with Ni Sepharose 6Fast Flow (GE) using the manufacturer's recommended protocol. HaloTag-β9(peptide 840) was purified using FPLC HisTrap FF 5 ml columns (Cytiva). Protein was eluted using a gradient elution up to 500 mM imidazole, dialyzed in PBS, and characterized to be >95% pure using SDS-PAGE. Proteins were stored in PBS + 50% glycerol at -20°C.

Calibrating the Luminescent Signal of the GloMax to the Handheld Luminometer.
A 4X solution containing recombinant Wuhan-Hu-1 nucleocapsid protein was prepared for a doseresponse curve and 150 μL was added to the jackets containing the master mix. A 30-fold dilution of Fz (Nano-Glo Live Cell Substrate; Promega N205) in PBSB was prepared and added at 300 μL/swab jacket for a final volume of 600 μL/swab jacket. Immediately after mixing, each reaction was split to allow for simultaneous reads in either a GloMax Discover Multimode Microplate Reader (Promega) or in the handheld luminometer. 200 μL of each reaction was transferred to a solid, white, nonbinding surface (NBS) 96-well plate (Costar) and read on a GloMax Discover Multimode Microplate Reader (Promega) collecting total luminescence using kinetic read over 1h. In parallel, the remaining 400 μL of each reaction remained in the swab jacket and was read with the handheld luminometer at 30min, 45min, and 1 h. To determine the correlation between the two instruments, the signals for each concentration of nucleocapsid protein were graphed and analyzed with linear regression.

Solution-Based SARS-CoV-2 RBD or N Immunoassay on a Handheld Luminometer.
A 4X master mix stock solution containing 120 ng/mL β9-labeled anti-SARS-CoV-2 spike RBD antibody D003, 240 ng/mL β10-labeled anti-SARS-CoV-2 spike RBD antibody D002, and 4 μM LgTrip protein was prepared in PBSB, and 100 μL/well was dispensed into the swab jacket. Either a 4X solution containing recombinant SARS-CoV-2 Spike RBD (refer to section 1.3 for inclusive list), heat-inactivated virus (refer to section 1.3 for inclusive list), or patient sample and 100 μL/ well was added to the swab jackets containing the master mix. Lastly, 200 μL/well of a 30-fold dilution of Fz (Nano-Glo Live Cell Substrate; Promega N205) in PBSB was added to the jackets. Assays were read on a standard handheld luminometer by collecting total luminescence using kinetic or endpoint reads, depending on the experimental design. The same method was carried out for the nucleocapsid model system by using 120 ng/mL β9labeled anti-SARS-CoV-2 N mAb Cat. 9547, 240 ng/mL β10-labeled anti-SARS-CoV-2 N mAb Cat. 9548, and 4 µM LgTrip and creating 4X titrations of recombinant CoV N proteins, heat-inactivated SARS-CoV-2 variants, or patient samples.

Testing the Effect of Transport Media on the SARS-CoV-2 N Immunoassay.
A 2X Master mix containing 60 ng/mL β9-labeled anti-SARS-CoV-2 N mAb Cat. 9547, 120 ng/mL β10-labeled anti-SARS-CoV-2 N mAb Cat. 9548, and 2 µM LgTrip in PBSB was made and 50 µL /well was added to a 96-well plate (Costar). A 2X 1:2 dilution series of various transport medias were spike with a constant 100 ng/ml SARS-CoV-2 N (Meridian Bioscience) in each dilution. This plate was incubated at ambient temperature for 90 min. After the incubation, 100 µL/well of 30-fold diluted Fz (Promega N205) was added and read on a GloMax.

Expression and Purification of β9 (Peptide 840) and β10 (HiBiT) Genetic Fusions with SARS-CoV-2 RBD Proteins.
HEK293/T cells were grown to approximately 70-90% confluency, then used to seed 2x 500mL spinner flasks with 1.25x10 8 cells and recommended complete media plus 5 mL 1X pluronic F-68 polyol. Flasks were incubated at 37°C, 5% CO2 with stirring at 60-80rpm for 2-4 hours before transfection of plasmids expressing IL6-β9-HaloTag Linker-RBD-3xFlag or IL6-β10-15Gly/Ser linker-3XFlag with a CMV promoter made into DNA:PEI complexes. The DNA:PEI complexes were prepared for each flask by adding 400 µg DNA (0.8 µg/mL) for each RBD genetic fusion with a His Tag to 25 mL serum free media (SFM). In a separate tube, 1.2 mL ViaFect was added to 25 mL SFM. Tubes were mixed separately for 5 min at room temperature at 750 rpm. The DNA/SFM was added dropwise to the PEI/SFM, mixed gently by pipetting, and incubated at room temperature for 20 min to ensure proper complex formation. After incubation, 50 mL transfection complex was added to the 450 mL of cell culture. Spinner flasks were grown for 48 h at 37°C, 5% CO2 with stirring at 60-80 rpm. Media was collected and genetic fusion proteins were batch purified with ANTI-FLAG M2 Affinity Gel (Sigma).

Complete SARS-CoV-2 Serology Assay Lyophilization in Swab Jackets and Characterization.
The 4X stock assay reagent solution contained the following: 2.5% w/v Pullulan, 5 mM 6-Aza2thiothymine (ATT), 5 mM ascorbic acid, 20 mM HEPES (pH 8.0), 90 mM Glycine, 20 mM Histidine, 25 mg/mL Sucrose, 0.01% Polysorbate 80, 240 ng/mL β10-labeled anti-SARS-CoV-2 RBD or N antibody (clone 505E 9A12 A3), 120 ng/mL β9-labeled anti-SARS-CoV-2 RBD or N antibody, 4 µM LgTrip 5146, and 40 µM Fz. All materials used to make the complete complex buffer were from Sigma. Aliquots (400 μL) were prepared in disposable swab jackets for lyophilization. The swab jackets were partially capped prior to loading into the lyophilizer (Virtis Genesis 12EL) with shelves pre-set to 4 °C. Product then underwent a freezing step with a shelf temperature of -50 °C for 2 h. Upon evacuation of the system the lyophilization process was performed between shelf temperatures of -25 °C and 25 °C and pressures of 75 and 200 mTorr. The ice sublimation phase lasted 8 h and the bound water desorption phase lasted 16 h. At the end of the lyophilization process, the swab jackets were covered with a temporary stopper under atmospheric conditions.

Human Subjects Nasopharyngeal Swab and Serum Sample Collection.
SARS-CoV-2 positive serum samples (n = 13) were obtained from local participants who previously had a SARS-CoV-2 PCR+ test result but were in the convalescent stage.  (n=34) of positive SARS-CoV-2 nasopharyngeal swabs were obtained and collected from the Wisconsin State Lab of Hygiene. Following HIPPA compliance, these samples were collected with informed consent from the volunteer, and samples were deidentified before use in this study.

Commercially Sourced Serum Samples.
Commercially available negative serum samples from pre-pandemic timepoints from healthy individuals were sourced from Boca Biolistics (n=12).

Protein Quantification.
Protein concentration was determined by measuring the absorbance at 280 nm with a spectrophotometer (NanoDrop 8000, Thermo Fisher), following the manufacturer's instructions.