. 2021 Dec 14;11:23921. doi: 10.1038/s41598-021-03154-6

Table 1.

Overview comparison of the Duke versus Monogram assays.

Component	Duke	Monogram
Purpose	Measures ability of antibody in a serum/plasma/monoclonal or purified antibody sample to neutralize SARS-CoV-2 pseudovirus
Principal	– SARS-CoV-2 pseudotyped virus expresses firefly luciferase upon infecting HEK 293 T cells, with relative light units (RLU) of luminescence directly proportional to the infectivity of the virus inoculum – Neutralizing antibodies inhibit pseudovirus infection of cells, resulting in lower RLUs – Serial dilutions of antibodies are used to construct a dose–response curve to quantify potency for each sample, recorded as 50% inhibitory dose (ID50) and 80% inhibitory dose (ID80)
Format and capacity	Performed in 96-well plate format and includes controls
Format and capacity	Up to 4 test samples in duplicate per plate	Up to 6 test samples in singlicate titrations per plate
Virus	Lentivirus:
Virus	Pseudotyped with SARS-CoV-2 G614 full-length Spike protein and packaged with TMPRSS2 serine protease and pHR' CMV Luc luciferase reporter genes	Pseudotyped with SARS-CoV-2 G614 full-length Spike protein and packaged with HIV genomic vector, pRTV1.FlucP.CNDO-∆U3, containing a luciferase reporter gene in place of HIV envelope
Cell Line	HEK 293 T cell line:
Cell Line	– Stably overexpressing the human ACE2 cell surface receptor protein (defined as 293 T/ACE2), – Cells may be used up to passage 46 or 4 months in culture	– Transiently transfected to express human ACE2 cell surface receptor protein along with a serine airway protease, TMPRSS2, for processing the S protein – Cells may be used up to passage 30 or as long as data maintains high quality
Controls	– Assay Plate Positive Control – anti-spike RBD mAb spiked into normal human serum. Included on every test plate – Assay Run Positive Controls – convalescent serum samples with high, medium, and low titers – Assay Run Negative Control – normal human serum (NHS). – Included on at least one plate in each assay run	– SARS-CoV-2 Positive Control (CPC#) – pool of COVID-19 convalescent plasma with positive SARS-CoV-2 neutralization titer. Included on every test plate – SARS-CoV-2 Negative Control (CNC#) – pool of COVID-19 convalescent plasma with negative SARS-CoV-2 neutralization titer. Included on every test plate – Daily Positive Controls – convalescent serum samples with high, medium, and low titers tested once per day – Specificity Control Pseudovirus – a lentiviral pseudovirus with Influenza H10N3 envelope tested with every sample and control to detect non-specific inhibition
Equipment	– 96-well Poly-L-Lysine or regular plates – GloMax Navigator Microplate Luminometer (Promega) – Manual liquid handling	– 96 well opaque Luminometer plates – Luminoskan Luminometers – Semi-automated liquid handling
Assay Range	1:10 to 1:781,250 or 1:20 to 1:1,562,500	1:40 to 1:787,320
Sample types	Heat-inactivated (56 °C, 30 min) serum (preferred), plasma, convalescent and vaccine incurred samples plus monoclonal antibodies and purified antibodies	Heat-inactivated (56 °C, 60 min) serum, plasma, convalescent and vaccine incurred samples plus monoclonal and purified antibodies
Basic steps	– Each sample is diluted serially five-fold in duplicate for a total of 8 dilutions starting at a 1:10 or 1:20 dilution (dilution after adding virus but before adding cells) – Virus is added to the plate (except cell control wells) and incubated for 45–90 min in humidified 37 °C and 5% CO₂ incubator – 293 T/ACE2 cells are harvested and 100 µl added to the plate at a density of 10,000 cells/well – Plates are incubated for 71–73 h in humidified 37 °C and 5% CO₂ incubator – Add Bright-Glo luciferase reagent to lysed cells and luminescence measured with a luminometer	– Each sample is diluted initially 1:20 and then diluted serially threefold to yield a total of 10 concentrations. The starting dilution of 1:20 becomes the final, reported as 1:40 after addition of the virus – Virus is added to the plate and incubated for 1–2 h in humidified 37 °C and 7% CO₂ incubator – Transfected 293/ACE2/TMPRSS2 cells are harvested and diluted to 10,000 cells/well and added to the plates – Plates are incubated for 3 days in humidified 37 °C and 7% CO₂ incubator – Steady Glo luciferase reagent is added to plate and luminescence measured with a luminometer
Calculations	Percent neutralization calculated as difference between RLUs of virus- and cell-only wells and test sample wells:
	$% N e u t r a l i z a t i o n = 100 % \times (1 - \frac{A v g . R L U (V i r u s + S a m p l e + C e l l s)}{A v g . R L U (V i r u s + D i l u e n t + C e l l s)})$ – Average RLU of cell control wells (without virus) are subtracted from RLUs of all other wells on a plate	$% N e u t r a l i z a t i o n = 100 % \times (1 - \frac{R L U (V i r u s + S a m p l e + C e l l s)}{A v g . R L U (V i r u s + D i l u e n t + C e l l s)})$
	ID50 and ID80 neutralizing antibody titers are expressed as the reciprocal of the sample dilution required to reduce RLU by 50% and 80%, respectively
Acceptance Criteria/ Plate level	– Positive Controls within acceptance range for ID50 or ID80 titers – Signal of virus-only wells is in range of RLU > 48,000 to < 1,000,000 – %CV ≤ 35% for virus only control wells	– Positive Control, CPC#, titer falls within expected range for the assay (± 3 SD of the mean) and within 30% of all CPC#s titers generated for the day – RLUs fall between ~ 10,000 and ~ 500,000
Acceptance Criteria/ Sample level	– Relative Percent Difference ≤ 30% between duplicate wells for each sample with ≥ 40% neutralization – Neutralization curves must cross 50% neutralization 0–1 time	– Percent inhibition values of serial dilutions must follow a logical progression, e.g. the 1:40 serum dilution should exhibit equal or more inhibition of viral infection than the 1:120 serum dilution. Once the data points decrease below maximum inhibition and are on the linear part of the curve, each succeeding data point (higher dilution) should be less inhibiting than the preceding point (lower dilution) – Graphs are rejected when antibody inhibition profiles (curves) have all or most (> 80%) of the points not falling on or within 20% of the extrapolated linear portion of the curve
Acceptance Criteria/ each Assay Run	ID50 or ID80 titers for Assay Run Positive and Negative Controls must be within acceptance range	Positive Control, CPC#, titer falls within expected range for the assay (± 3 SD of the mean) and within 30% of all CPC#s titers generated for the day
Validation Status	– Full validation for convalescent samples – Partial validation for vaccine incurred samples