A flow cytometry-based assay for serological detection of anti-spike antibodies in COVID-19 patients

Summary One of the key public health strategies in coronavirus 2019 disease (COVID-19) management is the early detection of infected individuals to limit the transmission. As a result, serological assays have been developed to complement PCR-based assays. Here, we report the development of a flow cytometry-based assay to detect antibodies against full-length SARS-CoV-2 spike protein (S protein) in patients with COVID-19. The assay is time-efficient and sensitive, being able to capture the wider repertoire of antibodies against the S protein. For complete details on the use and execution of this protocol, please refer to Goh et al. (2021).


Day 4
Step 5 Colony PCR Day 5 Step 6  Note: We have used 5 mg vector for digestion to ensure there is sufficient cleaved fragment to proceed to the next step. A lower vector DNA (such as 1-2 mg) can be used too.
Note: The amount of enzymes can be increased to a maximum of 10% of the total reaction volume. More than 10% might affect the digestion, due to the glycerol content.
Alternatives: XbaI and BamHI enzymes from other suppliers, such as Promega, (#R6181 and #R6021 respectively) can be used. b. Run the digest on 0.8% agarose TAE gel at 100 V for 90 min.
Note: Run 1 kb DNA marker.
Note: Run non-digested vector as a control. If the digest is not complete, the band profile will be similar to the control, with more bands in addition to the fragments of interest. In this case, set up the reaction with 0.5 mL more of each enzyme, or increase the enzyme volume to a maximum of 10% of the total reaction volume.
Note: The digest can be divided and run in 2-3 wells to allow better resolution on the gel. c. Gel-extract the vector backbone ($7.6 kb), using the NEB's Monarch gel extraction kit.
Alternatives: Other gel extraction kits can be used, such as QIAquick Gel Extraction Kit (-QIAGEN #28704). d. Quantify the DNA using a spectrophotometer. e. Store at À20 C until use.

OPEN ACCESS
Note: We advise to first calculate the amount of ligation reactions intended for Step 3a. If the amount of gel-extracted DNA falls below the calculated amount, repeat the enzymatic digest and gel-extraction.
2. Day 2: Preparation of the insert (encoding the S protein) a. Double-digest the insert with XbaI and BamHI for 2 h at 37 C, as described in step 1a.
Note: The chemically synthesized insert (by Genscript) is designed to be flanked by XbaI at the 5 0 end and BamHI at the 3 0 end. b. Run the digest on 0.8% agarose TAE gel at 100 V for 90 min. c. Gel-extract the insert fragment ($3.8 kb), as described in step 1c. d. Quantify the DNA using a spectrophotometer. e. Store at À20 C until use.
3. Day 3: Ligation of insert fragment into vector backbone a. Set up the ligation reaction as below: b. Incubate for 5-20 min at 20 C-22 C.

Note:
The ligation can also be incubated at 16 C for 12-16 h.
Note: In parallel, set a ligation negative control reaction, where only the digested vector is included and no insert is included. The double-digested vector has incompatible ends, hence ligation should not be possible.
4. Transformation of ligation mix into chemically competent bacterial cells. a. Add 2.5 mL ligation mix to 25 mL XL10-gold competent cells. b. Transform according to the manufacturer's instructions. c. Plate the mixture on LB-ampicillin agar plates (100 mg/mL ampicillin). d. Incubate the LB-ampicillin agar plates at 37 C for 12-16 h.
Note: Alternatively, other competent cells with low recombination capacity can be used such as top10 (Thermo Fisher Scientific #C404010).
Note: There should be no colonies on the plate transformed with the ligation negative control reaction, where only the vector is included. Colonies on this plate would mean either there is inefficient digestion or inefficient gel extraction (possibly because the digested fragments have not been resolved well on the gel. In this case, repeat step 1).
5. Day 4: perform a colony PCR to screen bacterial colonies containing the plasmid with insert, using SPseqF4 and IRESrev primers (Table 1)  Alternatives: Other polymerases, such AmpliTaq polymerase (Thermo Fisher Scientific Cat# N8080153) can also be used. b. Use a micropipette tip to touch the colony, dab onto a LB-ampicillin agar plate (100 mg/mL ampicillin, Sigma-Aldrich Cat# A0166) and then mix in the PCR reaction mix for each tube.
Note: Ensure that the picked colonies on the LB-ampicillin agar plate are numbered. c. Perform the PCR with the below cycling conditions: d. Analyze the colony PCR by running a 1% agarose TAE gel. A band of approximately 600 bp should be present if the insert is successfully cloned into the vector. e. Pick 3-5 positive colonies, each colony into 3-5 mL LB-ampicillin broth. Incubate on shaking (250 rpm) at 37 C for 12-16 h.
Note: Incubation should be no longer than 16 h as the colonies might be overgrown, affecting the DNA recovery.
Alternatives: Other plasmid extraction kits, such NucleoSpin Plasmid Mini kit (Macherey Nagel Cat# 740588.50) can also be used.
Note: The extraction of the plasmid can be scaled up by extracting from a 100 mL culture, using a QIAGEN plasmid Maxi kit (#12162). b. Sequence extracted plasmid using primers in Table 1.

Transfection to generate lentiviral particles
Timing: 4 days HEK293T cells are transfected to generate lentiviral particles.
Note: The culture medium for HEK293T cells is DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.
Step Cycle Temperature Time Note: The lentiviral particles are generated, using transfer plasmids and the pMD2.G, pRSV-Rev and pMDLg/pRRE packaging system. This is a 3rd generation, 4-plasmid system. 7. Day 1: Seed 0.5 3 10 6 HEK293T cells in 2 mL culture media into each well in 6 well plate.
Note: The cells should be 70-80% confluent the next day (before transfection).
Note: The cell number and transfection protocol below can be scaled by a factor of 0.4 if 12 well plate is used or by a factor of 2.5 if a 6 cm dish is used. Note: The transduction protocol described has been optimized using HEK293T cells. It has also been similarly applied to HEK293, EL4 and K562 cells. However, do ensure that all samples, that are going to be compared, are analyzed using the same cell line, as different cell lines might have different glycosylation modifications of the spike protein, affecting the antibody binding.
Note: The cells should be 70-80% confluent the next day (before transduction).
Note: Always include a negative control (a well where fresh culture media is added in place of the viral supernatant).
Note: It is recommended to determine the viral titer by qRT-PCR or p24 ELISA before transduction as different production lots might have different yields of virions. We have found that, if the transgene is with a phenotype detectable by flow cytometry (such as eGFP in this case), it is a better method of quantifying the viral titer than p24 ELISA or qRT-PCR (both of which measure incomplete/non-functional virus components in addition to functional virions). If it is the first time performing transduction, it is advisable to perform a few conditions by varying the amount of viral supernatant per well: eg. 2 mL, 20 mL, 200 mL. b. Add the polybrene/viral supernatant mixture to the well. c. Spin at 1200 3 g for 1 h at 32 C.
Note: Pre-warm the centrifuge to 32 C before use. d. Incubate at 37 C. e. At the end of the day ($7-8 h), aspirate out the infection medium and add fresh culture media to the cells. Continue incubation at 37 C for 48 h.

Day 1
Step 10 Seeding of cells

Day 2
Step 11 Transduction Medium change at the end of the day Day 4 Step 12  2. Seed 0.15 3 10 6 cells into each well in 96 V-bottomed well plates. a. All samples are analyzed in technical duplicates. b. Pellet the cells down by centrifugation at 300 3 g for 5 min.
3. Re-suspend cells in diluted plasma/serum samples. a. Dilute the samples at 1:100 in FACS buffer (10% FBS diluted in PBS) prior to addition to cells. b. Ensure that negative and positive control samples are also included. Eg. Anti-spike monoclonal antibody (e.g., Thermo Fisher Scientific #703958) can be used as positive controls and healthy control plasma/sera can be used as negative controls. 4. Incubate at 4 C for 30 min in the dark. 5. Wash twice with PBS by centrifugation at 300 3 g for 5 min. 6. Re-suspend cells in diluted secondary antibody incubation.
a. Dilute the secondary antibodies at 1:600 in FACS buffer prior to addition to cells. b. For IgG, IgM and IgA detection, the secondary antibody is anti-human IgG, anti-human IgM and anti-human IgA Alexa Fluor 647 antibodies in FACS buffer with 1 mg/mL propidium iodide. c. For IgG subclasses detection, the secondary antibody is mouse anti-human IgG1, IgG2, IgG3 and IgG4 antibodies in FACS buffer.
Note: Other fluorophores, other than Alexa Fluor 647, can also be used. One other possible option is Alexa Fluor 405, which have little compensation issues with the GFP-positive cells and the propidium iodide staining. We have chosen Alexa Fluor 647 as there is also little compensation issues with the GFP-positive cells and the propidium iodide staining.
Note: In place of propidium iodide, DAPI can also be used for staining to differentiate live/ dead cells. Alternatively, other live/dead viability dyes may be used.
7. Incubate at 4 C for 30 min in the dark. 8. Wash twice with PBS by centrifugation at 300 3 g for 5 min. 9. For IgG and IgM detection, add 100 mL FACS buffer to the well. Re-suspend well and analyze by flow cytometry. 10. For IgG subclasses detection, re-suspend cells in diluted tertiary antibody incubation.
a. Dilute the secondary antibodies at 1:600 in FACS buffer prior to addition to cells. b. The tertiary antibody is anti-mouse Alexa Fluor 647 antibodies in FACS buffer with 1 mg/mL propidium iodide (PI; Sigma-Aldrich #P4170). 11. Incubate at 4 C for 30 min in the dark. 12. Wash twice with PBS by centrifugation at 300 3 g for 5 min. 13. Add 100 mL FACS buffer to the well. Re-suspend well and analyze by flow cytometry.
a. Cells were gated on: (1) FSC-A/SSC-A to exclude cell debris (Figure 2A), (2) FSC-A/FSC-H to select for single cells ( Figure 2B), (3) FSC-A/PI to select for live cells (PI-negative population, Figure 2C), (4) FITC/Alexa Fluor 647 ( Figures 2D-2H). Binding is determined by the percentage of GFP-positive S protein-expressing cells that are bound by specific antibody, indicated by the events that are Alexa Fluor 647-and FITC-positive (Gate 2). A sample is defined as positive when the binding is more than mean + 3SD of the healthy controls.
Note: Cells are read on LSR4 laser (BD Biosciences), however, the cells can be read on any other cytometers with the following specifications (Table 3).

EXPECTED OUTCOMES
Using this assay, we are able to analyze the S protein-specific antibody profile of symptomatic and asymptomatic COVID-19 patients (Goh et al., 2021). While the antibody levels are lower in asymptomatic patients, the assay is highly sensitive and detects 97% of the asymptomatic infections. We also found that IgG1 is the dominant IgG subclass in both symptomatic and asymptomatic patients.

QUANTIFICATION AND STATISTICAL ANALYSIS
Quantification of S protein antibody by flow cytometry Specific antibody binding to cells was determined by LSRII 4 laser (BD Biosciences) and analyzed using FlowJo (Tree Star). Binding is determined by the percentage of GFP-positive S protein-expressing cells that are bound by specific antibody, indicated by the events that are Alexa Fluor 647-and FITC-positive (Gate 2).
2. Define a sample as positive when the binding is more than mean + 3SD of the healthy control individuals. The thresholds using the healthy control readings is based on the normal-like distribution of the healthy control reading where a mean + 3SD threshold would mean that there is less than a 0.13% chance of a false positive.
Note: In Goh et al. (Goh et al., 2021), our sample size of healthy control individuals was 22 and the Receiver Operating Characteristic (ROC) curves were constructed from each of the antibody binding with the healthy control individuals and SARS-CoV-2 patients as the true negatives and true positives respectively using the pROC library in R version 3.6.4.

LIMITATIONS
Similar to all serological assays, the risk of false positive diagnosis is one of the limitations of the assay. However, the assay consists of seven tests (IgM, IgA, IgG, and four IgG subclasses), allowing internal validation. Nevertheless, borderline positive results should be interpreted with caution. One other limitation of the SFB assay is the need for advanced planning. The assay is a cell-based assay, hence the dependence on cell culture requires careful planning ahead to ensure sufficient cell count. This limits the application of the assay for HTS. We suggest performing different serological assays in parallel: (1) this would complement each other to provide better diagnosis, and (2) other serological assays that allows high throughput screening application, could serve as the first round of screening, and the more sensitive SFB assay could provide confirmation and further investigation of borderline/ discrepant samples. As the SFB assay is a cell-based FACS assay, the acquisition of the samples can time-costly, especially when the sample size is large.

TROUBLESHOOTING
Problem 1 Inefficient digest of vector backbone (step 1 of before you begin).

Potential solution
Set up the digest reaction with 10 U of enzymes in excess per 5 mg vector.

Problem 2
No colonies following DNA ligation (step 3 and 4 of before you begin).

Potential solution
The DNA ligation can be optimized by: Incubating the ligation reaction at 16 C for 12-16 h. Ensuring efficient digest of the vector backbone and insert.

Problem 3
Low viral titer (step 9 of before you begin).

Problem 4
Insufficient cells for acquisition on the cytometer (step 13 of step-by-step method details).

Potential solution
Possibly due to significant cell loss throughout the assay. In this case, increase the cell number per well to 0.25 3 10 6 cell per well.

Problem 5
No binding, as indicated by absence of Alexa Fluor 647 staining (step 13 of step-by-step method details).

Potential solution
Possibly because the secondary or tertiary antibodies was left out. In this case, re-stain with secondary antibody incubation. Ensure that positive control samples are included.

Materials availability
All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability
This study did not generate any datasets/code.