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[Preprint]. 2024 Apr 2:2024.03.18.585599.
doi: 10.1101/2024.03.18.585599.

Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots

Katherine Berman  1 , Greta Van Slyke  1 , Hayley Novak  1 , Jean M Rock  1 , Rachel Bievenue  1 , Amanda K Damjanovic  1 , Kate L DeRosa  1 , Gianna Mirabile  1 , Roxie C Girardin  1 , Alan P Dupuis  1 , Kathleen A McDonough  1 , Monica M Parker  1 , Linda M Styer  1 , Nicholas J Mantis  1
Affiliations

Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots

Katherine Berman et al. bioRxiv. .

Update in

  • Quantitating SARS-CoV-2 neutralizing antibodies from human dried blood spots.
    Berman K, Van Slyke G, Novak H, Rock JM, Bievenue R, Damjanovic AK, DeRosa KL, Mirabile G, Phipps K, Machowski J, Bialosuknia S, Giradin RC, Dupuis AP, Payne AF, Lee WT, McDonough KA, Parker MM, Styer LM, Mantis NJ. Berman K, et al. Microbiol Spectr. 2024 Oct 29;12(12):e0084624. doi: 10.1128/spectrum.00846-24. Online ahead of print. Microbiol Spectr. 2024. PMID: 39470282 Free PMC article.

Abstract

Background: In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest.

Methods: Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay and a commercial real-time PCR-based inhibition assay (NAB-Sure™).

Results: DBS eluates from convalescent individuals were compatible with the spike-ACE2 inhibition assays, but not cell-based pseudovirus assays or PRNT. However, the insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers.

Conclusion: SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.

Keywords: Antibody; COVID-19; Human; Neutralizing; Serology.

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Conflict of interest statement

Conflicts of Interests (COI). The authors have no conflicts of interest to declare.

Figures

Figure 1.
Figure 1.. Generation of contrived DBS eluates.
(Top) Schematic showing generation of contrived DBS (cDBS), DBS punches, and eluate collection. (Bottom) Summary of Panels D, E, and H samples used in this study. RT = room temperature, TBS = tris buffered saline
Figure 2.
Figure 2.. Correlation between PRNT and MIA with cDBS eluates.
(Top) Workflow for the 8-plex MIA. (Bottom) Subset of Panel D cDBS eluates (n=15) underwent testing via 8-plex MIA and were compared to paired serum tested by PRNT. Pearson correlation of MFI values and SARS-CoV-2 neutralizing activity (%) is shown for the following antigens: (A) FLT; (B) FLS; (C) S1; (D) RBD; (E) sum spike (see text for details); and (F) N. The Pearson r value and p-values are shown as insets within each panel.
Figure 3.
Figure 3.. Compatibility of cDBS eluates with RVP neutralizing assay.
(Top) Workflow for the RVP neutralizing assay. (Bottom) (A) Neutralizing titers associated with CC 12.3 with IC50 shown as text. (B) Correlation between PRNT and RVP with matched Panel D serum samples; (C) Correlation between PRNT (% neutralization) generated with Panel D serum versus RVP generated from matched cDBS eluates; (D) Comparison of RVP neutralization of Panel D and Panel E cDBS eluates during serial dilutions. Asterisks indicate a significant difference between groups by 2-way ANOVA, where *p=0.02, **p=0.009, ***p=0.0002, ****p<0.0001. For (B) and (C), Pearson r and p-value are shown as a panel inset.
Figure 4.
Figure 4.. Correlation between Luminex-based ACE2 inhibition and SARS-CoV-2 neutralization activity.
(Top) Workflow for the ACE2 inhibition (iACE2) assay. (A) Examination of iACE2 activity in Panel D cDBS eluates (n=15) versus Panel E eluates (n=7). In (A), asterisks indicate a significant difference between groups by Welch’s t-test, where ***p=0.0006. (B) Correlation of iACE2 values from Panel D cDBS eluates, as shown in (A), to SARS-CoV-2 neutralizing activity (%) from paired serum analyzed via PRNT. cDBS eluates were evaluated in triplicate. The Pearson r and p-value are shown as a panel inset for (B). The dotted line on (B) represents the average iACE2 activity for SARS-CoV-2 negative cohort (Panel E; n=7).
Figure 5.
Figure 5.. NAB-Sure exhibits high concordance with PRNT.
(Top) Workflow for the NAB-Sure™ assay. (Panel A) Pearson correlation of NAB-Sure ™ NT50 from authentic DBS sample eluates compared to PRNT paired serum. DBS samples were evaluated in duplicate. (Panels B-D) Pearson correlation comparing NAB-Sure ™ NT50 for cDBS eluates and paired serum NT50 from PRNT for 14 Panel D samples perfomed in triplicate with each replicate shown. For A-D, Pearson r and p values are in panel insets.
Figure 6.
Figure 6.. Assessment of SAR-CoV-2 neutralizing titers in cDBS eluates from vaccinated individuals (Panel H).
(A) Panel H, D and E cDBS eluates were rank ordered from high to low (left to right) based on RBDWT MFI determined in the 8-plex MIA. (B) Correlation plot between Panel H RBDWT MFI and Diasorin Liaison values provided from Access Biologics LLC. Panel H cDBS eluate activities in (C) Luminex-based iACE2 assay, (E) RVP neutralization assay and (G) NAB-Sure ™ with corresponding correlation plots shown in right panels (D, F, H). Pearson r and p values are shown as a panel insets. As noted in the text, the Panel H eluates shown in Panel A were diluted 1:10 for the assays descried in panels B-H to avoid saturating MFI values that confound correlation analysis. Moreover, two of the highest Panel H eluates were diluted 1:2 prior to the NAbSure assay to achieve NT50 values.
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