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With IVD Medical Instrument to Do SARS-CoV-2 Neutralizing Antibody Rapid Test Kit

Neutralizing Antibody Fluerescent Test Kit & Neutralizing Antibody Rapid Test

The pseudovirus-neutralizing antibody assay was performed as previously described (5). Briefly, Vero cells were seeded in 96-well plates to produce a monolayer at the time of infection. Pretitrated amounts of rVSV-SARS-Cov-2 [phCMV3-SARS-CoV-2 spike SARS-CoV-2-pseduotyped VSV-ΔG-GFP (green fluorescent protein) were generated by transfecting HEK293T cells, ATCC CRL-3216] were incubated with serially diluted human plasma at 37°C for 1 hour before adding to confluent Vero cell monolayers (ATCC CCL-81) in 96-well plates. Cells were incubated for 12 to 16 hours at 37°C in 5% CO2. Cells were then fixed in 4% paraformaldehyde, stained with 1 μg/ml Hoechst, and imaged using a CellInsight CX5 imager to quantify the total number of cells expressing GFP. Infection was normalized to the average number of cells infected with rVSV-SARS-CoV-2 incubated with normal human plasma. The LOD was established as <1:20 on the basis of plasma samples from a series of unexposed control subjects. Negative signals were set to 1:19. Neutralization IC50 (median inhibitory concentration) titers were calculated using One-Site Fit LogIC50 regression in GraphPad Prism 8.0.

A high-throughput fluorescence-based neutralization assay

To fill in the gap for COVID-19 serodiagnosis and vaccine evaluation, we developed a fluorescence-based assay that rapidly and reliably measures neutralization of a reporter SARS-CoV-2 by antibodies from patient specimens. The assay was built on a stable mNeonGreen (mNG) SARS-CoV-2 where the mNG gene was engineered at the ORF7 of the viral genome (Fig. 1a)10. The complete sequence of mNG SARS-CoV-2 is described in Supplementary Fig. 1. Figure 1b depicts the flowchart of the reporter neutralization assay in a 96-well format. The assay protocol is detailed in Supplementary Methods. Briefly, patient sera were serially diluted and incubated with the reporter virus. After incubation at 37°C for 1 h, Vero CC-81 cells (pre-seeded in a 96-well plate) were infected with the virus/serum mixtures at a multiplicity of infection (MOI) of 0.5. At 16 h post-infection, the mNG-positive cells were quantitated using a high-content imaging reader (Fig. 1b). It should be noted that Vero CC-81 cells, not Vero E6 cells, were chosen for the mNG assay to enable the accurate quantification of fluorescent cells. Sixty COVID-19 serum specimens from RT-PCR-confirmed patients and 60 non-COVID-19 serum samples (archived before COVID-19 emergence) were analyzed using the reporter virus. For some COVID-19-positive specimens, the sample collection days post-viral RT-PCR positive were available and are indicated in Table ​Table1.1. After reporter viral infection, the cells turned green in the absence of serum (Fig. 1c, bottom panel); in contrast, incubation of the reporter virus with COVID-19 patient serum decreased the number of fluorescent cells (top panel). A dose-response curve was obtained between the number of fluorescent cells and the fold of serum dilution (Fig. 1d and Supplementary Fig. 2), which allowed for the determination of the dilution fold that neutralized 50% of fluorescent cells (NT50). The reporter assay rapidly diagnosed 120 specimens within 24 h: all 60 COVID-19 sera (specimens 1–60) showed positive NT50 of 35 to 5711, and all 60 non-COVID-19 sera (specimens 61–120) showed negative NT50 of <20 (Table 1).

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