Elisa 96 well plates, by Corning - Product details

1

Cardiolipin Binding Assay for HA-DV Variants

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ELISA 96-well plates (Costar, Corning, NY, USA) were coated with 50 μL per well of cardiolipin (Sigma, St. Louis, MO, USA) prepared at 200 μg/mL in ethanol and blocked for 2 h with 4% BSA in a 20 mM Tris, 100 mM NaCl buffer at pH 7.4. To generate a binding curve, increasing concentrations of HA-DV were applied to wells. The binding data were fit to a one-site model using the Gnuplot 5.0 program (http://www.gnuplot.info/). cardiolipin binding by HA-DV variants was compared to cardiolipin binding by HA-DV at protein concentrations of 500 nM and 1000 nM. Bound HA-tagged proteins were detected with HRP-conjugated anti-HA-tag antibody (ab1265, Abcam, Cambridge, MA, USA) using a TMB (3,3′,5,5′-tetramethylbenzidine) substrate. Absorbances at 450 nm were measured on a Spectramax 340PC Microplate Reader (Molecular Devices Inc., Sunnyvale, CA, USA).

Kolyada A., Barrios D.A, & Beglova N. (2017). Dimerized Domain V of Beta2-Glycoprotein I Is Sufficient to Upregulate Procoagulant Activity in PMA-Treated U937 Monocytes and Require Intact Residues in Two Phospholipid-Binding Loops. Antibodies, 6(2), 8.

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Cardiolipin Binding Assay for HA-DV Variants

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ELISA 96-well plates (Costar, Corning, NY, USA) were coated with 50 μL per well of cardiolipin (Sigma, St. Louis, MO, USA) prepared at 200 μg/mL in ethanol and blocked for 2 h with 4% BSA in a 20 mM Tris, 100 mM NaCl buffer at pH 7.4. To generate a binding curve, increasing concentrations of HA-DV were applied to wells. The binding data were fit to a one-site model using the Gnuplot 5.0 program (http://www.gnuplot.info/). cardiolipin binding by HA-DV variants was compared to cardiolipin binding by HA-DV at protein concentrations of 500 nM and 1000 nM. Bound HA-tagged proteins were detected with HRP-conjugated anti-HA-tag antibody (ab1265, Abcam, Cambridge, MA, USA) using a TMB (3,3′,5,5′-tetramethylbenzidine) substrate. Absorbances at 450 nm were measured on a Spectramax 340PC Microplate Reader (Molecular Devices Inc., Sunnyvale, CA, USA).

Kolyada A., Barrios D.A, & Beglova N. (2017). Dimerized Domain V of Beta2-Glycoprotein I Is Sufficient to Upregulate Procoagulant Activity in PMA-Treated U937 Monocytes and Require Intact Residues in Two Phospholipid-Binding Loops. Antibodies, 6(2), 8.

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3

Antibody Avidity Assessment Against SARS-CoV-2 Spike Protein

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Antibody avidity determination was performed essentially as reported previously [50 (link)]. ELISA 96-well plates (Costar, Corning, NY, USA) were used to immobilize the SARS-CoV-2 S1-S2-HIS ectodomain at 100 ng/well in carbonate buffer (50 mM NaHCO₃/Na₂CO₃, pH 9.6) at 4 °C overnight. After blocking in 2% MPBST, mouse sera were pooled to a final dilution of 1:300 in 2% MPBST and incubated for 1 h at 37 °C on the immobilized S1-S2-HIS. After washing with PBS with 0.05% Tween 20 (v/v) (PBST), the plates were incubated with the indicated dilutions of NaSCN for 15 min at room temperature and 350 rpm and then immediately washed with PBST. IgGs were detected using anti-mouse serum conjugated with HRP (A0168, Sigma–Aldrich, Munich, Germany), and the absorbance was measured as described above. The values obtained in the absence of NaSCN were normalized to represent 100% IgG binding. Hence, the avidity of spike-specific antibodies was calculated from the ratio of the absorbance of antibodies bound after treatment with graded concentrations of NaSCN relative to the signal in the absence of NaSCN. One-way ANOVA to compare multiple groups was performed with Dunnett’s correction for multiple analyses.

Kim Y., Zheng X., Eschke K., Chaudhry M.Z., Bertoglio F., Tomić A., Krmpotić A., Hoffmann M., Bar-On Y., Boehme J., Bruder D., Ebensen T., Brunotte L., Ludwig S., Messerle M., Guzman C., Mandelboim O., Hust M., Pöhlmann S., Jonjić S, & Čičin-Šain L. (2022). MCMV-based vaccine vectors expressing full-length viral proteins provide long-term humoral immune protection upon a single-shot vaccination. Cellular and Molecular Immunology, 19(2), 234-244.

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4

Sera Blockade of Antibody Binding to RBD

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Sera blockade of antibody binding was performed as described in Piccoli
et al.^{15 (link)}. Briefly, human IgG1
antibodies were biotinylated using the EZ-link NHS-PEO solid phase biotinylation
kit (Pierce). Each labeled antibody was tested for binding to RBD by ELISA, and
a concentration for each antibody competition experiment was selected to achieve
80% maximal binding (EC₈₀). ELISA 96-well plates (Corning) were
pre-coated overnight at 4°C with 1 μg/mL of mouse Fc-tagged RBD
antigen (Sino Biological) in PBS. Unlabeled sera/plasma were serially diluted
and added to ELISA plates for 30 min, followed by addition of biotinylated
anti-RBD antibody at its EC₈₀ concentration. After 30 min incubation,
plates were washed and antibody binding was detected using alkaline
phosphatase-conjugated streptavidin (Jackson ImmunoResearch). Plates were
washed, pNPP substrate (Sigma-Aldrich) was added, and plates were read at 405
nm. The percentage of inhibition of antibody binding was calculated as:
(1–(OD_sample − OD_{neg ctrl}) / (OD_pos
ctrl − OD_{neg ctrl}) × 100.

Starr T.N., Czudnochowski N., Liu Z., Zatta F., Park Y.J., Addetia A., Pinto D., Beltramello M., Hernandez P., Greaney A.J., Marzi R., Glass W.G., Zhang I., Dingens A.S., Bowen J.E., Tortorici M.A., Walls A.C., Wojcechowskyj J.A., De Marco A., Rosen L.E., Zhou J., Montiel-Ruiz M., Kaiser H., Dillen J., Tucker H., Bassi J., Silacci-Fregni C., Housley M.P., di Iulio J., Lombardo G., Agostini M., Sprugasci N., Culap K., Jaconi S., Meury M., Dellota E., Abdelnabi R., Foo S.Y., Cameroni E., Stumpf S., Croll T.I., Nix J.C., Havenar-Daughton C., Piccoli L., Benigni F., Neyts J., Telenti A., Lempp F.A., Pizzuto M.S., Chodera J.D., Hebner C.M., Virgin H.W., Whelan S.P., Veesler D., Corti D., Bloom J.D, & Snell G. (2021). SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape. Nature, 597(7874), 97-102.

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5

RBD-Binding IgG1 mAb Inhibition Assay

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Human anti-RBD full IgG1 mAbs were biotinylated using the EZ-Link NHS-PEO solid phase biotinylation kit (Pierce). Labeled mAbs were tested for binding to RBD by ELISA and the optimal concentration of each mAb to achieve 80% maximal binding was determined. Unlabeled mAbs or sera/plasma were serially diluted and added to ELISA 96-well plates (Corning) pre-coated overnight at 4°C with 1 μg/mL of RBD mouse Fc-tagged antigen (Sino Biological) in PBS. After 30 min, biotinylated anti-RBD mAbs were added at the concentration achieving 80% maximal binding and the mixture was incubated at room temperature for 230 min. Plates were washed and mAb binding was revealed using alkaline phosphatase-comjugated streptavidin (Jackson ImmunoResearch). After washing, pNPP substrate (Sigma-Aldrich) was added and plates were read at 405 nm. The percentage of inhibition was calculated as follow: (1−(OD sample−OD neg ctr)/ (OD pos ctr−OD neg ctr)]) × 100.

Piccoli L., Park Y.J., Tortorici M.A., Czudnochowski N., Walls A.C., Beltramello M., Silacci-Fregni C., Pinto D., Rosen L.E., Bowen J.E., Acton O.J., Jaconi S., Guarino B., Minola A., Zatta F., Sprugasci N., Bassi J., Peter A., De Marco A., Nix J.C., Mele F., Jovic S., Rodriguez B.F., Gupta S.V., Jin F., Piumatti G., Lo Presti G., Pellanda A.F., Biggiogero M., Tarkowski M., Pizzuto M.S., Cameroni E., Havenar-Daughton C., Smithey M., Hong D., Lepori V., Albanese E., Ceschi A., Bernasconi E., Elzi L., Ferrari P., Garzoni C., Riva A., Snell G., Sallusto F., Fink K., Virgin H.W., Lanzavecchia A., Corti D, & Veesler D. (2020). Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology. Cell, 183(4), 1024-1042.e21.

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6

SARS-CoV-2 Antibody Detection Protocol

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ELISA 96-well plates (Corning, NY, USA) were pre-coated overnight at +4 °C with 100 μL/well of RBD in PBS solution (200 ng/well), or ACE2 in PBS solution (300 ng/well), or ACE2 in 100 mM sodium carbonate-bicarbonate buffer (pH 9.6) (100 μL/well). SARS-CoV-2 NP was used for plate coating at 100 ng/well in PBS. Plates were washed with PBS—0.02% Tween (PBST) thrice, blocked with 250 μL/well of 3% BSA-PBS, washed with PBST and used immediately or sealed with the adhesive film and stored at –18 °C.
The analyzed sera were diluted with the 1% BSA-PBS as 1:2000 (RBD antigen) or as 1:5000 (NP antigen) and incubated in plate wells for 1 h at 37 °C. Wells were washed three times with PBST, secondary anti-human IgG antibody-HRP conjugate (Xema Co., Ltd., Moscow, Russia, cat. T271X@1702) was used at the 1:20000 dilution, the incubation time was 1 h at +37 °C.
Positivity indices (OD/CO) were calculated as the ratio of the mean optical density for the test sample and the mean optical density in negative wells plus three times the standard deviation, according to the [23 (link)].

Kolesov D.E., Sinegubova M.V., Dayanova L.K., Dolzhikova I.V., Vorobiev I.I, & Orlova N.A. (2022). Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD. Diagnostics, 12(2), 393.

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7

Competitive Binding Assay for RBD

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Unlabeled mAbs or plasma/sera were serially diluted, mixed with RBD mouse Fc-tagged antigen (Sino Biological, final concentration 20 ng/mL) and incubated for 30 min at 37°C. The mix was added for 30 min to ELISA 96-well plates (Corning) pre-coated overnight at 4°C with 2 μg/mL human ACE2 in PBS. Plates were washed and RBD binding was revealed using secondary goat anti-mouse IgG (Southern Biotech). After washing, pNPP substrate was added and plates were read at 405 nm. The percentage of inhibition was calculated as follow: (1−(OD sample−OD neg ctr)/(OD pos ctr−OD neg ctr)]) × 100.

Piccoli L., Park Y.J., Tortorici M.A., Czudnochowski N., Walls A.C., Beltramello M., Silacci-Fregni C., Pinto D., Rosen L.E., Bowen J.E., Acton O.J., Jaconi S., Guarino B., Minola A., Zatta F., Sprugasci N., Bassi J., Peter A., De Marco A., Nix J.C., Mele F., Jovic S., Rodriguez B.F., Gupta S.V., Jin F., Piumatti G., Lo Presti G., Pellanda A.F., Biggiogero M., Tarkowski M., Pizzuto M.S., Cameroni E., Havenar-Daughton C., Smithey M., Hong D., Lepori V., Albanese E., Ceschi A., Bernasconi E., Elzi L., Ferrari P., Garzoni C., Riva A., Snell G., Sallusto F., Fink K., Virgin H.W., Lanzavecchia A., Corti D, & Veesler D. (2020). Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology. Cell, 183(4), 1024-1042.e21.

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8

RBD-hACE2 Binding Inhibition Assay

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Blockade of WT RBD binding to hACE2 was performed, as previously described (Piccoli et al., 2020 (link)). Unlabeled mAbs were serially diluted, mixed with RBD mouse Fc-tagged antigen (Sino Biological, final concentration 20 ng/mL) and incubated for 30 min at 37°C. The mix was added for 30 min to ELISA 96-well plates (Corning) pre-coated overnight at 4°C with 2 μg/mL hACE2 in PBS. Plates were washed (EL406 washer/dispenser BSL2 M, Biotek) and RBD binding was revealed using a secondary goat anti-mouse IgG (Southern Biotech). After washing, pNPP substrate was added and plates were read at 405 nm (Synergy H1 Hybrid Multi-Mode plate reader, Biotek). The percentage of inhibition was calculated as follow: (1−(OD sample−OD neg ctr)/(OD pos ctr−OD neg ctr)) × 100.

Thomson E.C., Rosen L.E., Shepherd J.G., Spreafico R., da Silva Filipe A., Wojcechowskyj J.A., Davis C., Piccoli L., Pascall D.J., Dillen J., Lytras S., Czudnochowski N., Shah R., Meury M., Jesudason N., De Marco A., Li K., Bassi J., O’Toole A., Pinto D., Colquhoun R.M., Culap K., Jackson B., Zatta F., Rambaut A., Jaconi S., Sreenu V.B., Nix J., Zhang I., Jarrett R.F., Glass W.G., Beltramello M., Nomikou K., Pizzuto M., Tong L., Cameroni E., Croll T.I., Johnson N., Di Iulio J., Wickenhagen A., Ceschi A., Harbison A.M., Mair D., Ferrari P., Smollett K., Sallusto F., Carmichael S., Garzoni C., Nichols J., Galli M., Hughes J., Riva A., Ho A., Schiuma M., Semple M.G., Openshaw P.J., Fadda E., Baillie J.K., Chodera J.D., Rihn S.J., Lycett S.J., Virgin H.W., Telenti A., Corti D., Robertson D.L, & Snell G. (2021). Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity. Cell, 184(5), 1171-1187.e20.

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9

ACE2-RBD Binding Inhibition ELISA

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ACE2 blockade ELISA was performed as described by Piccoli et
al.^{15 (link)}. Unlabeled
antibodies were serially diluted, mixed with RBD mouse Fc-tagged antigen (Sino
Biological, final concentration 20 ng/mL) and incubated for 30 min at
37°C. The mix was added for 30 min to ELISA 96-well plates (Corning)
pre-coated overnight at 4°C with 2 μg/mL human ACE2 in PBS. Plates
were washed and RBD binding was revealed using secondary goat anti-mouse IgG
(Southern Biotech 1030–04). After washing, pNPP substrate was added and
plates were read at 405 nm. The percentage of inhibition was calculated as: (1
− (OD sample − OD neg ctrl)/(OD pos ctrl − OD neg ctrl)])
× 100.

Starr T.N., Czudnochowski N., Liu Z., Zatta F., Park Y.J., Addetia A., Pinto D., Beltramello M., Hernandez P., Greaney A.J., Marzi R., Glass W.G., Zhang I., Dingens A.S., Bowen J.E., Tortorici M.A., Walls A.C., Wojcechowskyj J.A., De Marco A., Rosen L.E., Zhou J., Montiel-Ruiz M., Kaiser H., Dillen J., Tucker H., Bassi J., Silacci-Fregni C., Housley M.P., di Iulio J., Lombardo G., Agostini M., Sprugasci N., Culap K., Jaconi S., Meury M., Dellota E., Abdelnabi R., Foo S.Y., Cameroni E., Stumpf S., Croll T.I., Nix J.C., Havenar-Daughton C., Piccoli L., Benigni F., Neyts J., Telenti A., Lempp F.A., Pizzuto M.S., Chodera J.D., Hebner C.M., Virgin H.W., Whelan S.P., Veesler D., Corti D., Bloom J.D, & Snell G. (2021). SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape. Nature, 597(7874), 97-102.

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Biotinylated anti-SARS-CoV-2 spike antibodies

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Human anti-S monoclonal antibodies (S2V29 for RBD site Ia, SA55 for RBD site IIa, S309 for RBD site IV^{23 (link)}, S3H3 for domain C/SD1^{60 (link)} and S2P6 for the stem helix^{61 (link)}) were biotinylated using the EZ-Link NHS-PEO solid phase biotinylation kit (Pierce). Labelled monoclonal antibodies were tested for binding to Wu-G614, BQ.1.1 and XBB.1 S by ELISA and the optimal concentration of each monoclonal antibody to achieve 80% maximal binding was determined. Plasma samples were serially diluted and added to ELISA 96-well plates (Corning) pre-coated overnight at 4 °C with 1 µg ml⁻¹ of S (Acrobiosystems) in PBS. After 30 min, biotinylated anti-S monoclonal antibodies were added at the concentration achieving 80% maximal binding and the mixture was incubated at room temperature for 30 min. Plates were washed and antibody binding was revealed using alkaline phosphatase-comjugated streptavidin (Jackson ImmunoResearch). After washing, pNPP substrate (Sigma-Aldrich) was added and plates were read at 405 nm. The percentage of inhibition was calculated as follow: (1 − (absorbance of sample − absorbance of negative control)/(absorbance of positive control − absorbance of negative control)) × 100.

Addetia A., Piccoli L., Case J.B., Park Y.J., Beltramello M., Guarino B., Dang H., de Melo G.D., Pinto D., Sprouse K., Scheaffer S.M., Bassi J., Silacci-Fregni C., Muoio F., Dini M., Vincenzetti L., Acosta R., Johnson D., Subramanian S., Saliba C., Giurdanella M., Lombardo G., Leoni G., Culap K., McAlister C., Rajesh A., Dellota E J.r., Zhou J., Farhat N., Bohan D., Noack J., Chen A., Lempp F.A., Quispe J., Kergoat L., Larrous F., Cameroni E., Whitener B., Giannini O., Cippà P., Ceschi A., Ferrari P., Franzetti-Pellanda A., Biggiogero M., Garzoni C., Zappi S., Bernasconi L., Kim M.J., Rosen L.E., Schnell G., Czudnochowski N., Benigni F., Franko N., Logue J.K., Yoshiyama C., Stewart C., Chu H., Bourhy H., Schmid M.A., Purcell L.A., Snell G., Lanzavecchia A., Diamond M.S., Corti D, & Veesler D. (2023). Neutralization, effector function and immune imprinting of Omicron variants. Nature, 621(7979), 592-601.

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Elisa 96 well plates

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16 protocols using elisa 96 well plates

Cardiolipin Binding Assay for HA-DV Variants

Cardiolipin Binding Assay for HA-DV Variants

Antibody Avidity Assessment Against SARS-CoV-2 Spike Protein

Sera Blockade of Antibody Binding to RBD

RBD-Binding IgG1 mAb Inhibition Assay

SARS-CoV-2 Antibody Detection Protocol

Competitive Binding Assay for RBD

RBD-hACE2 Binding Inhibition Assay

ACE2-RBD Binding Inhibition ELISA

Biotinylated anti-SARS-CoV-2 spike antibodies

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