Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection

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Plasmid construction

Plasmids were constructed by standard molecular biology methods. The DNA fragments of Spike-RBD, N protein, ACE2 and LgBiT were synthesized by IDT Technologies. The SmBiT tag was generated by overlap extension PCR. The Spike-RBD-5/15/25aa-LgBiT-12xHisTag, Spike-RBD-15/25aa-SmBiT-12xHisTag, N protein(44–180)-10aa-LgBiT-12xHisTag, N protein(44–180)-10aa-SmBiT-12xHisTag, LgBiT-10aa-N protein(44–257)-12xHisTag and SmBiT-10aa-N protein(44–257)-12xHisTag were generated by subcloning into a pFUSE-12xHisTag vector (adapted from the pFUSE-hIgG1-Fc vector from InvivoGen). The ACE2-Fc fusion plasmids were generated by subcloning the gene fragments of ACE2 and mutant into the pFUSE-hIgG1-Fc vector. The C004, C105 and C135 IgG LC and HC plasmids were a generous gift from the Nussenzweig lab (Rockefeller University). The CR3022 IgG plasmids were a generous gift from the Kim lab (Stanford University) and the Wilson lab (Scripps Research Institute). The C135 Fab was cloned by removing the Fc domain from the HC plasmid. SnapGene and ApE were used for cloning design and sequence verification.

Expression and protein purification

All proteins were expressed and purified from Expi293 BirA cells according to established protocols from the manufacturer (Thermo Fisher Scientific). Briefly, 30 μg of pFUSE (InvivoGen) vector encoding the protein of interest was transiently transfected into 75 million Expi293 BirA cells using the ExpiFectamine kit (Thermo Fisher Scientific). For the IgG and Fab proteins, 15 μg of each chain was transfected. Enhancer was added 20 h after transfection. Cells were incubated for a total of 3 d at 37 °C in an 8% CO2 environment before the supernatants were harvested by centrifugation. Fc-fusion proteins were purified by protein A affinity chromatography, and His-tagged proteins were purified by Ni-NTA affinity chromatography. Purity and integrity were assessed by SDS/PAGE. Purified protein was buffer exchanged into PBS and stored at −80 °C in aliquots. Concentration was verified by absorbance on a NanoDrop with NanoDrop 2000c software.

Solution serology protocol for in vitro, serum, blood, and saliva samples

LgBiT and SmBiT sensors for either the Spike or N protein were prepared at a final concentration of each sensor at 2 nM in PBS + 0.05% Tween-20 + 0.2% BSA (PBSTB). For in vitro IgGs or ACE2-Fc, the samples were prepared at 1:10 dilutions in PBSTB unless otherwise specified. Serum and blood samples were diluted to 1:12.5 for both the S and N sensor samples in PBSTB unless otherwise specified. Healthy individual saliva was spiked in with CR3022 and used undiluted or diluted 1:2 in PBSTB. Then, 10 μl of the 2 nM sensor mix and 10 μl of the sample were combined in a 384 Lumitrac white plate (Greiner), skipping every other well and row to avoid potential bleedover in signal. The plate was mixed on a plate shaker for 20 min. NanoLuc substrate was diluted according to protocol 1:50 in NanoLuc dilution buffer (Promega), and 15 μl was added to each well, followed by a 10-min incubation period for the signal to stabilize. Luminescence was measured on a Tecan M200 infinite plate reader with an integration time of 1,000 ms. Tecan i-control plate reader software was used for data acquisition.

Competition serology protocol for in vitro and serum samples

The competition serology assay was performed similarly to the solution serology assay except that the S sensors were individually pre-incubated at 4 nM with 4 μM of C004 Fab, C105 Fab or C135 Fab for the in vitro competition assay and C135 Fab only for the serum competition assay. The two sensors + Fab were combined 1:1 to make a 2 nM mix, and 10 μl of this mix was added to the assay as described above.

Epitope binning experiment

Biolayer interferometry data were measured using an Octet RED384 (ForteBio). Biotinylated Spike RBD protein was immobilized on the streptavidin biosensor (ForteBio). After blocking with biotin, the sensor was loaded with one IgG followed by another IgG or ACE2-Fc to determine epitope binning. PBS with 0.05% Tween-20 and 0.2% BSA was used for all diluents and buffers. ForteBio Octet Data Acquisition software was used for acquiring data, and ForteBio Octet Data Analysis software was used for analyzing data.

Spike protein ELISA assay

The Spike ELISA assay was performed as previously described. Briefly, 384 MaxiSorp plates were coated with 100 μl of 0.5 μg ml−1 NeutrAvidin for 1 h. The plate was washed three times with PBS + 0.05% Tween-20 (PBST) followed by incubation with 20 nM S-RBD for 30 min. After three washes, the plate was blocked with 3% non-fat milk in PBS for 1 h. The plate was washed three times before the addition of 1:50 dilutions of serum in 1% non-fat milk for 1 h. After three washes, secondary anti-human Fab (Jackson ImmunoResearch Laboratories, 109-036-097 (1:5,000)), anti-human IgM (Sigma-Aldrich, A6907 (1:3,000)) or anti-human IgG (Sigma-Aldrich, A0170 (1:3,000)) antibody was added and incubated for 30 min before the addition of TMB for 3 min. The reaction was quenched with 1 M phosphoric acid, and absorbance was read on a Tecan M200 infinite plate reader at 450 nm.

Inter-day and intra-day assay with liquid dispensers

Plasma was diluted 12.5-fold into a 96-well plate, and, subsequently, 10 µl was dispensed into an individual flat-bottom white plate (Greiner Lumitrac 200, 384-well plates) using a Biomek FXP Automated Workstation (Beckmann Coulter). Next, 10 µl of 1 nM biosensors was dispensed using Thermo Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific) to assay plates and incubated at room temperature for 20 min. Then, 15 µl of substrate was added using the same reagent dispenser and incubated at room temperature for 10 min. Luminescence was read on a Tecan M200 infinite plate reader with an integration time of 1,000 ms.

Simulated automation of the spLUC assay on a robotic platform

To facilitate high-throughput screening of serum, a semi-automated approach was developed and simulated using the UCSF Antibiome Center robotics platform40. Serum in 96-well plates is first diluted 12.5-fold, and 10 µl is dispensed into four individual flat-bottom white plates (Greiner Lumitrac 200, 384-well plates) using a Biomek FXP Automated Workstation (Beckmann Coulter). Serum-containing assay plates are then transferred to a robotics protocol with dispensing of biosensor and substrate followed by luminescence reading. Although one iteration of 96 samples takes 40 min, each additional iteration takes an additional 3.5 min, limited by luminescence reading (1 s per well plus plate transfer). As such, it is estimated that 40 plates (3,840 assays) could be run in 3 h. The robotics run was developed and simulated using Thermo Momentum software (v5.0.6).

Lyophilization of sensors and dilution buffer

The S and N protein sensors were flash frozen in liquid nitrogen at concentrations between 10 μM and 60 μM in 10 μl. The dilution buffer was frozen in liquid nitrogen in 5-ml aliquots. A small hole was poked into the caps of the samples and left on a BenchTop K (VirTis) lyophilizer overnight. The next day, the sensors were reconstituted in 10 μl of double-distilled water (ddH2O), and concentration was verified by NanoDrop. The dilution buffer was similarly reconstituted in 5 ml of ddH2O.

Vacuum-dried centrifugation of substrate sample

Next, 20 μl of substrate was aliquoted in a dark Eppendorf tube and subjected to vacuum centrifugation overnight on a Genevac instrument. The substrate was stored in the dark for 2 d at room temperature before reconstitution in 20 μl of 100% methanol. The substrate was diluted 1:50 as normal in dilution buffer for the assay.

Serum, plasma, whole blood and saliva samples

The initial small patient cohort was a generous gift from the Wilson lab (UCSF) and heat inactivated at 56 °C for 1 h before storage at −80 °C. The first (outpatient) sample serum set (Cohort 1) was a generous gift from the Wilson lab (UCSF) and the Nussenzweig lab (Rockefeller University). These samples were heat inactivated at 56 °C for 1 h and stored at 4 °C in a 1:1 dilution in 40% glycerol, 40 mM HEPES (pH 7.3) and 0.04% NaN3 in PBS. The second (inpatient) sample serum set (Cohort 2) was a generous gift from the Wang lab (Stanford University) and was stored at −80 °C as pure serum samples. The third plasma cohort (Cohort 3) and blood samples were generous gifts from the Greenhouse lab (UCSF) and the Henrich Lab (UCSF) as part of the LIINC study. The plasma samples were stored at 4 °C in a 1:1 dilution in 40% glycerol, 40 mM HEPES (pH 7.3) and 0.04% NaN3 in PBS. The whole blood was stored undiluted at 4 °C. Healthy blood samples were purchased from Vitalant and stored undiluted at 4 °C. The saliva samples were obtained as unstimulated, unexpectorated saliva and were stored at −80 °C. Before being assayed, the samples were thawed and centrifuged at 9,000g to remove any insoluble or coagulated matter. Control saliva from November 2019 was purchased from Lee Biosolutions, stored at −20 °C and processed similarly.

Data and statistical analysis

All graphing and statistical analysis were performed in GraphPad Prism or Microsoft Excel. The unpaired multiple t-test was performed in Prism to compare conditions in each patient for the competition spLUC assay. The non-parametric Spearman correlation analysis was used in Prism to determine the correlation R value between datasets. When two groups were compared, an unpaired Mann–Whitney test was performed to determine the difference between datasets. For comparison of three or more groups, a Kruskal–Wallis test with Dunn’s multiple comparison post hoc testing was used. A two-tailed P value was used to determine statistical significance for all analysis. P < 0.05 was considered statistically significant.

Ethics declaration

All patient samples were obtained under protocols approved by the institutional review boards (IRBs) and in accordance with the Declaration of Helsinki. Samples were de-identified before delivery to the lab where all assays described here were performed. Cohort 1 samples were a kind gift from M. Nussenzweig, M. Caskey and C. Gaebler of Rockefeller University, collected under Rockefeller IRB protocol DRO-1006. Cohort 2 samples from Kaiser Permanente were collected under Stanford University IRB protocol 55718. Cohort 3 samples were collected under UCSF IRB protocol 20-30479. Influenza virus vaccination samples were from a US cohort enrolled at the Rockefeller University Hospital in New York City in 2012–2013 under a protocol approved by the IRB of Rockefeller University (protocol TWA-0804). Samples from people with seasonal coronavirus infections were collected at the University of Chicago. Samples were de-identified serums of healthcare workers who had respiratory illnesses, were swabbed and tested positive for common cold coronavirus infections in 2019 (University of Chicago protocol 09-043-A).

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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