Immunoassay-Compatible Inactivation of SARS-CoV-2 in Plasma Samples for Enhanced Handling Safety

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) inactivation is an important step toward enhanced biosafety in testing facilities and affords a reduction in the biocontainment level necessary for handling virus-positive biological specimens. Virus inactivation methods commonly employ heat, detergents, or combinations thereof. In this work, we address the dearth of information on the efficacy of SARS-CoV-2 inactivation procedures in plasma and their downstream impact on immunoassays. We evaluated the effects of heat (56 °C for 30 min), detergent (1-5% Triton X-100), and solvent-detergent (SD) combinations [0.3-1% tri-n-butyl phosphate (TNBP) and 1-2% Triton X-100] on 19 immunoassays across different assay formats. Treatments are deemed immunoassay-compatible when the average and range of percentage recovery (treated concentration relative to untreated concentration) lie between 90-110 and 80-120%, respectively. We show that SD treatment (0.3% TNBP/1% Triton-X100) is compatible with more than half of the downstream immunoassays tested and is effective in reducing SARS-CoV-2 infectivity in plasma to below detectable levels in plaque assays. This facile method offers enhanced safety for laboratory workers handling biological specimens in clinical and research settings.

Figure 1

Schematic workflow of the study experimental design. Figure elements were generated from Servier Medical Art templates licensed under Creative Commons Attribution 3.0 Unported License (https://smart.servier.com) and Microsoft Office art tools.

Figure 2

Morphological effects on Vero cells after exposure to plasma at various concentrations. (A) Vero cells exposed for 1 h to neat and 2× diluted plasma displayed distinct morphological changes and possibly cell death and loss of substrate adherence. Residual cells appeared rounded up and shrunk in size. These cells were subsequently washed and cultured with fresh media. They appear to recover morphologically after 72 h. (B) Plasma was spiked with SARS-CoV-2 to an estimated titer of 1 × 10⁵ PFU/mL. Serial dilutions of spiked plasma were added to Vero cells for subsequent plaque assays. No plaques were observed in wells that were exposed to neat plasma.

Figure 3

Assessment of cytotoxicity of SD-treated plasma and its mitigation by filtration through Pierce detergent removal columns. (A) Microscopy images (1 h post-treatment) and (B) crystal violet staining (72 h post-treatment) of Vero cells exposed to column-filtered and nonfiltered SD-treated plasma. Cytotoxicity and morphological changes were observed after 1 h exposure to filtered neat plasma, and a fivefold dilution was sufficient to overcome the effects of residual SD in the sample. Cells recovered at 72 h post-treatment, as indicated by strong crystal violet staining in wells containing neat filtered plasma. Without SD removal, a minimum 100-fold dilution is required to overcome SD cytotoxicity. Negative controls are Vero cells without added plasma.

Figure 4

Virus recovery after plasma filtration through Pierce detergent removal columns. Plasma samples were spiked with SARS-CoV-2 to an estimated 1 × 10⁵ PFU/mL. Plaque assays were then performed in duplicate on plasma samples with and without filtration through Pierce detergent removal columns. Plaque counts were assessed at 10^–2 dilution, and the average virus titer (n = 2) of the unfiltered and filtered samples was found to be comparable at 1.4 × 10⁵ and 0.94 × 10⁵ PFU/mL, respectively.

Figure 5

SARS-CoV-2 plaque assays with SD removal. Following SD-treatment, the plasma samples were filtered through Pierce detergent removal columns prior to addition to Vero cells. Positive control plasma was also subjected to the same column filtration procedure. Clear plaques were observed at all dilutions except at 10^–5 for the positive control wells. In contrast, no plaques were observed at all dilutions post-SD treatment. Panel shows the results from one of three independent experiments.