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Fluosphere neutravidin conjugated beads

Manufactured by Thermo Fisher Scientific
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FluoSphere™ NeutrAvidin™-conjugated beads are fluorescent polymer microspheres conjugated with NeutrAvidin, a deglycosylated form of avidin. These beads can be used for a variety of bioassay applications that require specific and reversible binding to biotinylated molecules.

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Lab products found in correlation

Ez link sulfo nhs lc lc biotin, by Thermo Fisher Scientific (2 mentions) Gelatin veronal buffer, by Boston BioProducts (1 mentions) Lyophilized guinea pig complement, by Cedarlane (1 mentions) Cd66b, by BioLegend (1 mentions) Cd14 apc cy7, by BD (1 mentions) Cd66b pacific blue, by BioLegend (1 mentions) Ez link sulfo nhs lc lc, by Thermo Fisher Scientific (1 mentions)

3 protocols using fluosphere neutravidin conjugated beads

1

Antibody-Dependent Complement Deposition Assay

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ADCD was conducted as previously described.82 (link) Briefly, NVX-CoV2373 Spike protein was biotinylated using EZ-link™Sulfo-NHS-LC-LC-Biotin (Thermo Fisher), and then coupled to red FluoSphere™ NeutrAvidin™-conjugated beads (Thermo Fisher). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum (ADCD 1:10 dilution) for 2 h at 37°C, and then washed to remove unbound antibody. The immune complexes were then incubated with lyophilized guinea pig complement (Cedarlane) and diluted in gelatin veronal buffer with calcium and magnesium (Boston Bioproducts) for 30 min. C3 bound to immune complexes was detected by fluorescein-conjugated goat IgG fraction to guinea pig Complement C3 (MP Biomedicals). Flow cytometry was performed to identify the percentage of beads with bound C3. Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1) (Figure S1).
Gorman M.J., Patel N., Guebre-Xabier M., Zhu A.L., Atyeo C., Pullen K.M., Loos C., Goez-Gazi Y., Carrion R J.r., Tian J.H., Yuan D., Bowman K.A., Zhou B., Maciejewski S., McGrath M.E., Logue J., Frieman M.B., Montefiori D., Mann C., Schendel S., Amanat F., Krammer F., Saphire E.O., Lauffenburger D.A., Greene A.M., Portnoff A.D., Massare M.J., Ellingsworth L., Glenn G., Smith G, & Alter G. (2021). Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination. Cell Reports Medicine, 2(9), 100405.
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2

ADCP and ADNP Antibody Assays for NVX-CoV2373

Cited 1 time
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ADCP and ADNP were conducted as previously described.80 (link),81 (link) Briefly, NVX-CoV2373 Spike protein was biotinylated using EZ-link™Sulfo-NHS-LC-LC-Biotin (Thermo Fisher), and then coupled to yellow/green FluoSphere™ NeutrAvidin™-conjugated beads (Thermo Fisher, F8776). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum (ADNP 1:50 dilution, ADCP 1:100 dilution) for 2 h at 37°C, and then washed to remove unbound antibody. The immune complexes were then incubated overnight with THP-1 cells (ADCP), or for 1 h with RBC-lysed whole blood (ADNP). THP-1 cells were then washed and fixed in 4% PFA, while the RBC-lysed whole blood was washed, stained for CD66b+ (Biolegend) to identify neutrophils, and then fixed in 4% PFA. Flow cytometry was performed to identify the percentage of quantity of beads phagocytosed by THP-1 cells or neutrophils, and a phagocytosis score was calculated (% cells positive × Median Fluorescent Intensity of positive cells). Flow cytometry was performed with an iQue (IntelliCyt) or LSRII(BD) and analysis was performed using IntelliCyt ForeCyt (v8.1) or FlowJo V10.7.1 (Figure S1).
Gorman M.J., Patel N., Guebre-Xabier M., Zhu A.L., Atyeo C., Pullen K.M., Loos C., Goez-Gazi Y., Carrion R J.r., Tian J.H., Yuan D., Bowman K.A., Zhou B., Maciejewski S., McGrath M.E., Logue J., Frieman M.B., Montefiori D., Mann C., Schendel S., Amanat F., Krammer F., Saphire E.O., Lauffenburger D.A., Greene A.M., Portnoff A.D., Massare M.J., Ellingsworth L., Glenn G., Smith G, & Alter G. (2021). Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination. Cell Reports Medicine, 2(9), 100405.
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3

Antibody-Dependent Cellular Phagocytosis and Antibody-Dependent Neutrophil Phagocytosis Assays

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ADCP and ADNP were conducted as previously described [80 (link),81 (link)]. Briefly, spike or RBD proteins were biotinylated using EDC (Thermo Fisher Scientific) and EZ-link Sulfo-NHS-LC-LC (Thermo Fisher Scientific) and then coupled to yellow/green and then coupled to yellow/green FluoSphere NeutrAvidin-conjugated beads (Thermo Fisher Scientific, F8776). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum (ADNP 1:50 dilution, ADCP 1:100 dilution) for 2 hours at 37°C and then washed to remove unbound antibody. The immune complexes were then incubated overnight with THP-1 cells (ADCP), or for 1 hour with RBC-lyzed whole blood (ADNP). THP-1 cells were then washed and fixed in 4% PFA, while the RBC-lyzed whole blood was washed, stained for CD66b Pacific Blue (BioLegend - San Diego, California, USA), CD3-AlexaFluro700 (BD Biosciences - Miami, Florida, USA), and CD14-APC-Cy7 (BD Biosciences) to identify neutrophils (CD3- CD14- CD66b+) and then fixed in 4% PFA. Flow cytometry was performed to identify the percentage of quantity of beads phagocytosed by THP-1 cells or neutrophils, and a phagocytosis score was calculated (% cells positive × median fluorescent intensity of positive cells). Flow cytometry was performed with an LSRII (BD), and analysis was performed using FlowJo V10.7.1.
Zhu D.Y., Gorman M.J., Yuan D., Yu J., Mercado N.B., McMahan K., Borducchi E.N., Lifton M., Liu J., Nampanya F., Patel S., Peter L., Tostanoski L.H., Pessaint L., Van Ry A., Finneyfrock B., Velasco J., Teow E., Brown R., Cook A., Andersen H., Lewis M.G., Lauffenburger D.A., Barouch D.H, & Alter G. (2022). Defining the determinants of protection against SARS-CoV-2 infection and viral control in a dose-down Ad26.CoV2.S vaccine study in nonhuman primates. PLoS Biology, 20(5), e3001609.
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