Single‐reaction multi‐antigen serological test for comprehensive evaluation of SARS‐CoV‐2 patients by flow cytometry

Cáceres‐Martell, Yaiza; Fernández‐Soto, Daniel; Campos-Silva, Carmen; García‐Cuesta, Eva M.; Casasnovas, José M.; Navas-Herrera, David; Beneítez‐Martínez, Alexandra; Martínez‐Fleta, Pedro; Alfranca, Arántzazu; Sánchez‐Madrid, Francisco; Escudero-López, Gabriela; Vilches, Carlos; Jara‐Acevedo, Ricardo; Reyburn, Hugh; Rodrı́guez-Frade, José Miguel; Valés‐Gómez, Mar

doi:10.1002/eji.202149319

Cited by 11 publications

(6 citation statements)

References 22 publications

(31 reference statements)

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“…Higher epitope density likely allows for monogamous multivalent binding of low affinity antibodies. Other immunoassays to detect specific anti-Spike antibodies by flow cytometry have been developed and either do not show IgM detection ( 20 , 21 ) or they do efficiently detect IgM on beads where the antigen has been adsorbed ( 22 – 24 ) or on the surface of HEK-293T cells where the antigen is overexpressed ( 25 – 28 ). In these two approaches epitope density is higher compared to the Jurkat-Spike cells of the JFCI, in line with our argument.…”

Section: Discussionmentioning

confidence: 99%

Vaccine Type-, Age- and Past Infection-Dependence of the Humoral Response to SARS-CoV-2 Spike S Protein

Romero-Pinedo¹,

Quesada²,

Horndler

et al. 2022

Front. Immunol.

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The emergence of COVID-19 has led to a worldwide challenge for the rapid development of vaccines. Several types of safe and effective vaccines have been available in a time frame never seen before. Now that several hundred million people have been vaccinated there is an opportunity to compare vaccines in terms of protection and immune response. Here, we have applied a highly sensitive multiplexed flow cytometry method to measure simultaneously IgM, IgG1 and IgA anti-spike protein antibodies generated in response to three vaccines: ChAdOx1 (Oxford-AstraZeneca), mRNA-1273 (Moderna), and BNT162b2 (Pfizer-BioNTech). We have found that mRNA vaccines (mRNA-1273 and BNT162b2) induce a stronger humoral response, both after the first and the second dose, than the adenovirus-based ChAdOx1 vaccine. We also found that, in the elderly, antibody titers negatively correlate with the age of the donor but, also, that antibody titers remain stable for at least 6 months after complete vaccination. Finally, we found that one dose of BNT162b2 is sufficient to induce the highest antibody titers in seropositive pre-vaccination donors. We hope these data will help to guide future decisions on vaccination strategies.

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Section: Discussionmentioning

confidence: 99%

Vaccine Type-, Age- and Past Infection-Dependence of the Humoral Response to SARS-CoV-2 Spike S Protein

Romero-Pinedo¹,

Quesada²,

Horndler

et al. 2022

Front. Immunol.

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“…Purified Spike protein was prepared as described in [ 20 ]. Briefly, a recombinant cDNA cloned in pCDNA3.1 vector and coding for soluble S protein containing a T4 fibritin trimerization sequence, a Flag epitope and an 8xHistag (all at the C-terminus), was transfected in HEK293F cells using standard procedures.…”

Section: Methodsmentioning

confidence: 99%

SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells

Barreda

Santiago

Rodríguez³

et al. 2021

Cells

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Dendritic cells (DCs) are the most potent antigen-presenting cells, and their function is essential to configure adaptative immunity and avoid excessive inflammation. DCs are predicted to play a crucial role in the clinical evolution of the infection by the severe acute respiratory syndrome (SARS) coronavirus (CoV)-2. DCs interaction with the SARS-CoV-2 Spike protein, which mediates cell receptor binding and subsequent fusion of the viral particle with host cell, is a key step to induce effective immunity against this virus and in the S protein-based vaccination protocols. Here we evaluated human DCs in response to SARS-CoV-2 S protein, or to a fragment encompassing the receptor binding domain (RBD) challenge. Both proteins increased the expression of maturation markers, including MHC molecules and costimulatory receptors. DCs interaction with the SARS-CoV-2 S protein promotes activation of key signaling molecules involved in inflammation, including MAPK, AKT, STAT1, and NFκB, which correlates with the expression and secretion of distinctive proinflammatory cytokines. Differences in the expression of ACE2 along the differentiation of human monocytes to mature DCs and inter-donor were found. Our results show that SARS-CoV-2 S protein promotes inflammatory response and provides molecular links between individual variations and the degree of response against this virus.

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“…To the best of our knowledge, there is only three studies employing a cell‐free flow cytometric approach to evaluate the humoral immune response (Cáceres‐Martell et al . 2021 ; Dogan et al . 2021 ; Egia‐Mendikute et al .…”

Section: Resultsmentioning

confidence: 99%

“…These beads were used for simultaneous detection of IgG, IgM and IgA by flow cytometry, showing results with low background signals and high specificity and sensitivity (Cáceres‐Martell et al . 2021 ). However, magnetic beads are not compatible with certain flow cytometers.…”

Section: Resultsmentioning

confidence: 99%

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Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG, IgM and IgA using beads covalently coupled to the nucleocapsid protein

Zattoni

Huergo

Gerhardt

et al. 2022

Letters in Applied Microbiology

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Flow cytometry has emerged as a promising technique for detection of SARS‐CoV‐2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS‐CoV‐2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho‐SMCC chemistry. BUV395 anti‐IgG, BB515 anti‐IgM, biotinylated anti‐IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell‐free assay efficiently discriminate COVID‐19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5–96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry‐based diagnosis.

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Single‐reaction multi‐antigen serological test for comprehensive evaluation of SARS‐CoV‐2 patients by flow cytometry

Cited by 11 publications

References 22 publications

Vaccine Type-, Age- and Past Infection-Dependence of the Humoral Response to SARS-CoV-2 Spike S Protein

Vaccine Type-, Age- and Past Infection-Dependence of the Humoral Response to SARS-CoV-2 Spike S Protein

SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells

Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG, IgM and IgA using beads covalently coupled to the nucleocapsid protein

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