Lentiviral Pseudovirus Neutralization Assay for SARS-CoV-2 Spike-Specific Antibody Titration in HEK293T-ACE2 Cells Using a Luciferase Readout

Antibody/serum is tested in 8-point 3-fold serial dilutions (e.g., starting 1:20 for serum or 10 ug/mL for mAb), each in duplicate wells, against a fixed pseudovirus input normalized to ~1e5-1e6 relative light units (RLU) in virus-only controls. Each 96-well plate includes virus-only (no antibody, 100% infection) wells, cells-only (no virus, background) wells, a known neutralizing mAb as a positive control, and an irrelevant/isotype antibody as a negative control. Assays are run on 3 independent days. Bald (no-envelope) pseudovirus controls confirm entry is spike-dependent.

BSL-2. Although single-cycle and replication-defective, lentiviral pseudovirus is handled at BSL-2 in a Class II biosafety cabinet; treat the spike-pseudotyped particles as potentially infectious for one round. Use lab coat, gloves, eye protection. Decontaminate with 10% bleach (10 min) or 70% ethanol; bleach liquid waste to 1% final; autoclave solids. Avoid generating aerosols. Polybrene and luciferase reagents are irritants. IBC registration for lentiviral vectors and spike constructs is required; confirm no replication-competent lentivirus by p24/RCL testing for new producer lots.

Positive control: a characterized neutralizing mAb run in the same dilution series must yield its expected IC50 (within ~2-fold of historical). Negative control: isotype-matched non-neutralizing IgG and naive/pre-immune serum should show no neutralization (signal near virus-only). Virus-only wells define 0% neutralization (100% infection); cells-only wells define background to subtract. Bald-pseudovirus (no spike) control confirms ACE2/spike-dependent entry. A bare-vector pseudovirus (e.g., VSV-G) tests for non-specific serum cytotoxicity masquerading as neutralization.

Virus-only wells should read 1e5-1e6 RLU with a signal-to-background (vs cells-only) > 100. A neutralizing sample produces a clean sigmoid from ~100% infection at low antibody to <10% at high antibody, with a defined IC50/NT50. Potent mAbs give IC50 in the ng/mL range; convalescent/vaccine sera give NT50 from ~1:50 to >1:5000. Hill slopes near 1 indicate well-behaved single-site neutralization.

• HEK293T cells for pseudovirus production (ATCC CRL-3216)
• HEK293T-ACE2 stable target cells (ACE2-overexpressing)
• Lentiviral packaging plasmids: psPAX2 (gag/pol) and pHIV-Luc-ZsGreen or pLenti-Luc transfer vector
• SARS-CoV-2 spike expression plasmid (codon-optimized, D614G or variant of interest, with 19-aa cytoplasmic tail truncation)
• Transfection reagent: PEI (1 mg/mL) or Lipofectamine 3000
• DMEM + 10% FBS + 1x pen/strep growth medium
• ONE-Glo or Bright-Glo luciferase substrate
• Polybrene (hexadimethrine bromide), 8 ug/mL working
• Reference neutralizing mAb (positive control) and isotype IgG (negative control)
• White opaque 96-well tissue culture plates

To measure antibody-mediated neutralization of SARS-CoV-2 by titrating serum or monoclonal antibody against a spike-pseudotyped lentivirus encoding firefly luciferase, infecting ACE2-expressing HEK293T cells, and deriving the 50% neutralization titer (NT50) or IC50 from luminescence dose-response curves. This provides a quantitative, BSL-2-compatible correlate of protection for vaccine and therapeutic antibody evaluation.

1. Pseudovirus production (day 0): seed 6e6 HEK293T in a 10-cm dish. Next day co-transfect spike plasmid, psPAX2, and luc transfer vector (ratio ~1:1:1, total 15 ug) with PEI. Replace medium at 6-16 h.
2. Harvest supernatant at 48-72 h, clarify by 0.45 um filtration, aliquot, and store at -80 C. Titer on HEK293T-ACE2 to find the dilution giving ~1e5-1e6 RLU.
3. Seed HEK293T-ACE2 at 1e4 cells/well in white 96-well plates 24 h before infection (or co-seed at infection).
4. Prepare 8-point 3-fold antibody dilutions in 60 uL medium per well in a separate dilution plate.
5. Add 60 uL of pre-titered pseudovirus to each antibody dilution; include virus-only and cells-only wells. Incubate antibody+virus 1 h at 37 C.
6. Add the 120 uL antibody/virus mix onto target cells (with polybrene 8 ug/mL final). Incubate 48-72 h at 37 C, 5% CO2.
7. Equilibrate plates to room temperature, add 100 uL luciferase substrate per well, mix, incubate 3 min, and read luminescence on a plate luminometer (1 s/well integration).

Independent variable: antibody/serum concentration (8-point dilution series). Dependent variable: luciferase RLU and the derived percent neutralization, IC50/NT50. Controlled variables: pseudovirus input (normalized RLU), ACE2 target cell number (1e4/well), polybrene concentration, antibody-virus pre-incubation time (1 h), infection duration (48-72 h), and substrate/integration settings.

If a serum or antibody contains spike-specific neutralizing activity, then increasing antibody concentration will progressively block pseudovirus entry, producing a sigmoidal reduction in luciferase signal; the IC50/NT50 will correlate with live-virus PRNT50 within ~2-3 fold and discriminate neutralizing from non-neutralizing samples.

Subtract mean cells-only background from all wells. Percent neutralization = 100 x (1 - (RLUsample - background)/(RLUvirus-only - background)). Fit percent neutralization vs log10 concentration to a 4-parameter logistic (variable-slope) curve in GraphPad Prism or Python (scipy curve_fit). Report IC50 (mAb, ug/mL or ng/mL) or NT50 (serum, reciprocal dilution) with 95% CI. Flag fits with R2 < 0.9 or top/bottom plateaus outside 80-120%/0-20% for re-test.

1. Low virus-only RLU (<1e4): poor transfection or low ACE2 - re-titer pseudovirus, confirm ACE2 expression by anti-ACE2 flow, increase virus input. 2. High background in cells-only: substrate contamination or autoluminescence - use fresh substrate, white plates, and proper blanking. 3. No neutralization by the positive-control mAb: spike misfolded or cytoplasmic-tail issue - verify spike plasmid identity and use the 19-aa-truncated tail for higher incorporation. 4. Apparent neutralization with VSV-G control (non-specific): serum cytotoxicity/complement - heat-inactivate serum 56 C/30 min, lower top concentration. 5. Steep/shallow Hill slopes or poor fits: tighten dilution spacing around IC50, increase replicates.

Report IC50/NT50 as geometric mean +/- geometric SD across 3 independent assays. Compare two antibodies/groups by unpaired t-test on log10(IC50) (alpha=0.05); for >2 groups use one-way ANOVA with Tukey's correction. Correlation with live-virus PRNT uses Spearman's rho. Power: detecting a 3-fold IC50 difference with assay SD ~0.2 log10 at 80% power and alpha=0.05 requires n=3 independent runs per group.