For HDL and (V)LDL quantification, Human ApoA-I and ApoB Duplex ELISA kit (Cell Biolabs) was used according to manufacturer instructions. Samples and standards were measured in duplicate. For CD63 assay, anti-CD63 antibodies (4 μg/mL; see Table S1 for antibody information) were loaded (50 μL/well) in a 96 well-plate (Nunc MaxiSorp flat-bottom, Thermofisher) and incubated overnight at 4 °C. After washing twice with 200 μL of 0.1% BSA in PBS, plates were blocked with 1% BSA (200 μL/well) for 2 h at RT. After washing twice as described before, biotinylated anti-CD63 antibodies (500 ng/mL) were loaded (50 μL/well) and incubated for 1 h at RT. After washing twice, HRP-conjugated streptavidin (1:20,000 diluted in 0.1% BSA; #405210, BioLegend) were loaded (50 μL/well) and incubated for 20 min at RT. After washing three times, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB, BioLegend) were loaded per well and incubated for 30 min at RT. Reactions were stopped by adding 50 μL of stop solution, and absorbance was measured at 450 nm on a plate reader (Tecan). To estimate CD63-positive EV numbers, a calibration curve was generated using serially diluted EV samples whose EV numbers were counted by NTA (Figure S10
Nunc maxisorp flat bottom
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc MaxiSorp flat-bottom is a type of laboratory microplate. It is designed with a flat-bottom well shape.
Automatically generated - may contain errors
Lab products found in correlation
Golgistop, by BD (4 mentions)
Brefeldin a, by Merck Group (4 mentions)
Rosettesep, by STEMCELL (4 mentions)
Anti cd3 alexafluor700, by BD (4 mentions)
Anti ifn γ apc, by BD (3 mentions)
Anti cd56 pe cy7, by BD (3 mentions)
Anti mip1β pe, by BD (3 mentions)
Fix and perm a and b solutions, by Thermo Fisher Scientific (3 mentions)
Anti cd107a phycoerythrin pe cy5, by BD (2 mentions)
Anti cd16 allophycocyanin apc cy7, by BD (2 mentions)
Igg anti spike protein antibody, by Thermo Fisher Scientific (2 mentions)
Anti fd bacteriophage antibody, by Merck Group (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by BioLegend (1 mentions)
R35 72, by BD (1 mentions)
Protein g agarose resin, by Merck Group (1 mentions)
Anti mouse igg hrp antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugated streptavidin, by BioLegend (1 mentions)
Anti cd16 apc cy7, by BD (1 mentions)
Anti ifn γ fitc, by BD (1 mentions)
Anti cd107a pe cy5 stain, by BD (1 mentions)
32 protocols using nunc maxisorp flat bottom
1
Quantification of HDL, LDL, and CD63+ EVs
2
Quantification of OVA-specific IgE by ELISA
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The levels of OVA-specific IgE in sera were determined using ELISA. Briefly, 10-fold diluted serum (100 μl for each sample) was added to the wells of an OVA-coated ELISA plate (NuncMaxiSorp™ flat-bottom, Thermo Fisher Scientific), followed by addition of biotinylated rat anti-mouse IgE (R35-72, 2 μg/ml, BD Biosciences), horseradish peroxidase-conjugated avidin (40-fold dilution, BD Biosciences), and NeA-Blue (tetramethylbenzidine substrate; Clinical Science Products, Mansfield, MA, USA). The reaction was stopped by the addition of 3 N H3PO4, followed by measurement of the absorbance using a VersaMax™ ELISA reader (Molecular Devices, Temecula, CA, USA) at the wavelength of 450 nm, corrected to the absorbance at the wavelength of 540 nm.
3
Purification and ELISA Quantification of IgG and IgA
4
ELISA-Based NK Cell Activation Assay
5
SARS-CoV-2 Spike Protein Antibody ELISA
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ELISAs were performed in 96-microwell plates coated with 150 ng/well of SARS-CoV-2 Spike (aa 16 to 1,213) His-tagged recombinant protein (Thermo Fisher) and 1010 phage or AAVP particles/50 μL of phosphate-buffered saline (PBS) overnight at 4 °C (Nunc MaxiSorp flat bottom, Thermo Fisher Scientific). Coated plates were blocked with PBS containing 5% low-fat milk and 1% bovine serum albumin (BSA) for 1 h at 37 °C. Two-fold serial dilutions (starting at 1:32) of sera in blocking buffer were added to separate the wells and incubated for 1 to 2 h at 37 °C. Following three washes with PBS and PBS containing 0.1% of Tween-20, bound antibodies were detected with an anti-mouse IgG horseradish peroxidase (HRP)-conjugated (Jackson ImmunoResearch) at optical density (OD) at 450 nm. Commercially available polyclonal IgG anti-Spike protein antibody (Thermo Fisher, MA5-35949) or anti-fd bacteriophage antibody (Sigma Aldrich, B7786) served as positive controls.
6
Antibody Response to N-expressing Constructs
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To detect the potential of our N-expressing constructs to induce specific antibody responses in IFNα/β/γR−/− mice (before the challenge experiment), we developed an in-house enzyme linked immunosorbent assay (ELISA). Briefly, infected SW-13 cells with 1 moi of the Ank-2 strain were collected 2 days post-inoculation (dpi) and cell lysates were used as ELISA antigens. A total of 45 µg of the cell lysate was added to each well of a Nunc MaxiSorp flat-bottom (Thermo Fisher Scientific) in a bicarbonate buffer (pH 8) and incubated overnight at 4 °C. The next day, 1/1000 diluted serum samples (on day 28) from immunized IFNα/β/γR−/− mice were added to each well and incubated for 2 h at room temperature (RT). Subsequently, anti-mouse IgG-HRP antibody (Abcam, Cambridge, MA, USA) at a dilution of 1/10,000 was added and further incubated at RT for 1 h, followed by adding 3, 3′, 5, 5′-Tetramethylbenzidine (TMB ELISA Peroxidase) substrate. Finally, the reaction was stopped by adding 2N H2SO4. The results of the unvaccinated mice serum samples were subtracted from the cell lysate as background.
7
Enzyme-Linked Immunosorbent Assay Protocol
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Antibodies (4 μg/ml; see table S1 for antibody information) were loaded (50 μl per well) in a 96-well plate (Nunc MaxiSorp flat-bottom, Thermo Fisher Scientific) for adsorption (12 hours at 4°C). After washing twice with PBS (200 μl) containing 0.1% BSA, we treated the plate with 1% BSA (200 μl per well) for 2 hours at 20°C, washed it twice, and then loaded it with plasma samples (50 μl per well) for target capture (2 hours, 20°C). After washing twice as described above, we added biotinylated detection antibodies (500 ng/ml, 50 μl per well) for target labeling (1 hour at 20°C). After washing out excess antibodies, we added horseradish peroxidase–conjugated streptavidin (1:20,000 diluted in 0.1% BSA; #405210, BioLegend) to each well (50 μl), waited for 20 min (20°C), and washed the plate three times. Last, we added 3,3′,5,5′-tetramethylbenzidine (100 μl, BioLegend) to each well and let the mixture react (30 min at 20°C). We stopped the reaction by adding a stop solution (50 μl) and read optical absorbance at 450 nm (Tecan).
8
Standardized Rabies Virus Neutralization Assay
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In this study, the RFFIT protocol standardized at the KVAFSU-CVA Rabies Diagnostic Laboratory, WOAH Reference Laboratory for Rabies, Department of Veterinary Microbiology, Veterinary College, Bengaluru, was used for all serum samples [16 ,17 ]. The serum samples were incubated in a water bath for complement inactivation at 56 °C for 30 min. Later, the samples were serially diluted in a flat-bottomed 96-well microtiter plate (Nunc MaxiSorp™ flat-bottom, Thermo Fisher Scientific, Waltham, MA, USA). Following this, 100 TCID50 of rabies virus was added to all wells except cell controls, and then incubated at 37 °C for 90 min. Furthermore, approximately 25,000–30,000 BHK-21 cells/well were seeded into all wells of the plate and incubated at 37 °C for 48 h in a 5% CO2 incubator. After the incubation, the medium was decanted from the plate without disturbing the monolayer, and the cells were fixed with 70% chilled acetone for 30 min at −20 °C. The fixed cells were incubated with a 1:5 diluted fluorescein-labeled anti-RABV nucleoprotein antibody (Fujirebio Diagnostics, Malvern, PA, USA) for 1 h at 37 °C. The plates were examined using a fluorescent microscope and observed for fluorescent foci. The titer of RVNA was estimated in comparison with the WHO reference serum.
9
Flow Cytometric Analysis of Antibody-Dependent NK Cell Activation
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Natural killer cell (NK) activation and degranulation via CD107a, IFN-γ and MIP-1β detection was assessed via an ELISA-based antibody-dependent natural killer (NK) cell activation assay [52 (link)]. ELISA plates (Thermo Fisher NUNC MaxiSorp flat bottom) were coated with gp140 ConS (200 ng per well) at 37°C for 2 h followed by blocking with 5% BSA in PBS overnight at 4°C. NK cells were isolated from buffy coats from healthy donors with RosetteSep (Stem Cell Technologies) and rested over night with 1 ng/ml IL-15. The next day, after washing the blocked ELISA plates, 50 μl of samples at a 1:25 dilution in PBS were added to each well. Plates were incubated at 37°C for 2 h to allow immune complex formation. Then, 5×104 NK cells with anti-CD107a-PE-Cy5 stain (BD), brefeldin A (5 mg/ml) (Sigma), and GolgiStop (BD) were added to each well and incubated for 5 h at 37°C. NK cells were fixed and permeabilized using Perm A and B solutions (ThermoFisher). Cells were subsequently stained for surface markers with anti-CD16 APC-Cy7 (BD), anti-CD56 PE-Cy7 (BD) and anti-CD3 AlexaFluor 700 (BD). Intracellular staining included anti-IFNγ FITC (BD) and anti-MIP-1β PE (BD). Acquisition occurred by flow cytometry (IntelliCyt, iQue Screener plus). NK cells were defined as CD3- and CD56+. The antibody-dependent NK cell degranulation assay was performed in duplicate across two blood donors.
10
Antibody Titers in Vaccinated Mice
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Anti-rSAG1m and anti-rNbHsp90.3 antibodies in the sera of vaccinated mice were determined by ELISA. Briefly, 96-well microtiter plates (Nunc MaxiSorp™ flat-bottom, Thermo Fisher, Waltham, MA, USA) were coated overnight at 4 °C with 5 µg/ml of rSAG1m or 50 µg/ml of TLA (SAG1TLA). Serial dilutions of mice sera were carried out to determine the total IgG (IgGt) titer. The cut-off value was defined as the media of pre-immune sera absorbance values plus three standard deviations. For isotype determination, mice sera were diluted 1:100. Either goat anti-mouse IgG-horseradish peroxidase conjugated (1:5000) (Sigma-Aldrich, St. Louis, MO, USA), rat anti-mouse IgG1, IgG2a or IgG2b horseradish peroxidase conjugated (1:10000) (Sigma-Aldrich) were used as secondary antibodies. Tetramethyl-benzidine substrate (TMB; Invitrogen) was added and plates were read at 655 nm with an automatic ELISA reader (Synergy H1; Bio-Tek, Winooski, VT, USA).
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