To measure neutralizing antibody (NAb) activity in convalescent plasma, 5-fold serial dilutions of plasma were incubated for 1 h at 37°C in 96-well plates with an aliquot of HIV-1- or VSV-based SARS-CoV-2-pseudotyped virus containing approximately 1 × 103 infectious units. Thereafter, 100 μl of the plasma/virus mixture was added to target cells (293TAce2 cl.13 or Huh7.5 cells) in 96-well plates. Cells were cultured for 48 h (HIV-1 pseudotype viruses) or 16 h (VSV pseudotype viruses). Then, cells were washed twice and lysed and NanoLuc luciferase activity in lysates was measured using either the Nano-Glo luciferase assay system (Promega) and a Modulus II microplate multimode reader (Turner BioSystem) or a Glowmax Navigator luminometer (Promega). The 50% neutralizing titer (NT50) for plasma was determined using 4-parameter nonlinear regression in Prism 8.4 (GraphPad).
Glowmax navigator luminometer
Manufactured by Promega
The Glowmax Navigator luminometer is a compact, high-performance instrument designed for luminescence-based assays. It provides accurate and reliable detection of luminescent signals, making it suitable for a variety of laboratory applications.
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4 protocols using glowmax navigator luminometer
1
Measuring SARS-CoV-2 Neutralizing Antibodies
2
NanoLuc Luciferase Assay for Cell-based Experiments
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For the NanoLuc luciferase assays, cells were washed gently, twice with PBS and lysed in Lucifersase Cell culture Lysis reagent (Promega). NanoLuc luciferase activity in the lysates was measured using the Nano-Glo Luciferase Assay System (Promega) and a Modulus II Microplate Multimode reader (Turner BioSystem) or a Glowmax Navigator luminometer (Promega), as described previously (Schmidt et al., 2020 (link)). To record GFP+ cells, 12-well plates were photographed using an EVOS M7000 automated microscope. For flow cytometry, cells were trypsinized, fixed and enumerated using an Attune NxT flow cytometer. The half maximal inhibitory concentrations for plasma (NT50), and monoclonal antibodies (IC50) was calculated using 4-parameter nonlinear regression curve fit to raw or normalized infectivity data (GraphPad Prism). Top values were unconstrained, the bottom values were set to zero.
3
Quantifying SARS-CoV-2 Neutralization Assays
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For the NanoLuc luciferase assays, cells were washed twice, carefully, with PBS and lysed with 50 μl/well of Luciferase Cell Culture Lysis reagent (Promega). NanoLuc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (Promega). Specifically, 25 μl of substrate in NanoGlo buffer was mixed with 25 μl cell lysate in black flat bottom plates and incubated for 5 min at RT. NanoLuc luciferase activity was measured using a Modulus II Microplate Multimode reader (Turner BioSystem) or a Glowmax Navigator luminometer (Promega), using 0.1s integration time. Relative luminescence units (RLU) obtained were normalized to those derived from cells infected with SARS-CoV-2 pseudovirus in the absence of plasma/antibodies.
To record GFP+ cells, 96-well plates were photographed using and EVOS M7000 automated microscope. Alternatively, cells were trypsinized, fixed with 2% paraformaldehyde, washed and enumerated using an Attune NxT flow cytometer equipped with a 96-well autosampler.
The half maximal inhibitory concentration for plasma (NT50), antibodies (IC50) was determined using 4-parameter nonlinear regression curve fit to raw infectivity data measured as relative light units, or as the percentage of infected cells (GraphPad Prism). The top values were unconstrained, the bottom values were set to zero.
To record GFP+ cells, 96-well plates were photographed using and EVOS M7000 automated microscope. Alternatively, cells were trypsinized, fixed with 2% paraformaldehyde, washed and enumerated using an Attune NxT flow cytometer equipped with a 96-well autosampler.
The half maximal inhibitory concentration for plasma (NT50), antibodies (IC50) was determined using 4-parameter nonlinear regression curve fit to raw infectivity data measured as relative light units, or as the percentage of infected cells (GraphPad Prism). The top values were unconstrained, the bottom values were set to zero.
4
Neutralizing Antibody Assay for SARS-CoV-2
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To measure neutralizing antibody activity in convalescent plasma, five-fold serial dilutions of plasma were incubated for 1 hour at 37°C in 96-well plates with an aliquot of HIV-1 or VSV-based SARS-CoV-2 pseudotyped virus containing approximately 1×103 infectious units. Thereafter, 100 μl of the plasma/virus mixture was added to target cells (293TAce2 cl.13, or Huh7.5) cells in 96-well plates. Cells were cultured for 48h (HIV-1 pseudotype viruses) or 16h (VSV pseudotype viruses). Then, cells were washed twice, lysed and NanoLuc Luciferase activity in lysates was measured using either the Nano-Glo Luciferase Assay System (Promega) and a Modulus II Microplate Multimode reader (Turner BioSystem) or a Glowmax Navigator luminometer (Promega). The half maximal neutralizing titer (NT50) for plasma, was determined using a 4- parameter nonlinear regression in Prism 8.4 (GraphPad).
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