In brief, recombinant SARS- CoV-2 spike ectodomain protein or VZV gE protein (Genscript) diluted in coating buffer (Biolegend) were used to coat 96-well EIA/RIA plates (Greiner Bio-one, 100 ng per well) at 4 °C overnight. The plates were then washed with 1 PBS-T (0.05% Tween-20) and blocked with 2% BSA in PBS-T for 2 h at room temperature. Serum samples with serial dilutions were added and incubated for 2 h at room temperature. After washing, HRP-conjugated goat anti-mouse IgG antibody (1:10,000) was added and incubated for 1 h. TMB substrate (Invitrogen) was then used for colour development and the absorbance was read at 450 nm using BioTek microplate reader. End-point titres were calculated as the largest sample dilution factor yielding a signal that exceeds 2.1-fold value of the background58 (link).
Coating buffer
Manufactured by BioLegend
Sourced in United States
Coating buffer is a laboratory solution used to prepare surfaces for the immobilization of biomolecules, such as proteins or antibodies. It is a core component in various immunoassay and cell-based assay protocols.
Automatically generated - may contain errors
Lab products found in correlation
Nunc maxisorp 96 well elisa plate, by Thermo Fisher Scientific (2 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Avidin hrp, by BioLegend (1 mentions)
Stop solution, by BioLegend (1 mentions)
Ovalbumin (ova), by BioLegend (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sars cov 2 s protein, by ACROBiosystems (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Siinfekl peptide, by InvivoGen (1 mentions)
Igg2b, by Southern Biotech (1 mentions)
8 protocols using coating buffer
1
SARS-CoV-2 Spike Protein Antibody Assay
2
Quantifying Antigen-Specific IgE and IgG Levels
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Blood was collected from the rats’ tail veins. After centrifugation, the sera were collected and stored at −80°C until analyzed. The levels of OVA-specific IgE and IgG were measured by enzyme-linked immunosorbent assays (ELISA). In brief, Nunc™ MaxiSorp™ ELISA plates (BioLegend, San Diego, USA) were coated with 10 μg/ml OVA in coating buffer (BioLegend, San Diego, USA) overnight for OVA-specific IgE detection, 1 μg/ml for OVA-specific IgG detection. Serum samples (1/10 dilutions in Assay Diluent B, BioLegend) were added to the plates and incubated overnight at 4°C. Biotin-conjugated mouse anti-Rat IgE secondary antibody (1:2,000; Thermo Fisher Scientific, USA) or biotin-conjugated mouse anti-Rat IgG (H+L) secondary antibody (1:100,000; Thermo Fisher Scientific, USA) was separately added to the plates. Thereafter HRP avidin (BioLegend, San Diego, USA), diluted 1:2,000, was added. Plates were developed with tetramethylbenzidine (TMB) for 15 min at room temperature, stopped by the addition of Stop Solution (BioLegend), and read at 450 and 570 nm. The antibody levels were revealed from the observed magnitude difference between OD450 and OD570. Values less than 0.1 were regarded as undetectable and excluded in this assay.
3
Immobilized αCD19 FMC63 Antibody Assay
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Incubation of 12 well non-treated culture plates were done with 500 µL of coating buffer (BioLegend) containing 5 µg/mL of αCD19 FMC63 antibody and stored overnight at 4°C. The media was carefully removed and 1 mL of 5% BSA-PBS blocking solution was added. After 20 min of incubation at room temperature, the blocking solution was removed and 4×106 CAR T cells on 4 mL of RPMI supplemented with 100 IU/mL of IL-2 were added.
4
SARS-CoV-2 Antibody Quantification ELISA
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SPL Maxibinding ELISA plates (SPL) were coated with 33 ng of SARS-CoV-2 S protein (Acro-Biosystems) or SARS-CoV-2 receptor binding protein (RBD) (Sino Biological) in 100 µl (final concentration of 330 ng/ml) coating buffer (Biolegend) with overnight incubation at 4 °C. After routine wash and block with 2% Bovine Serum Albumin, (BSA) (Sigma–Aldrich), 100 µl of serial diluted mice or macaques serum were added to wells and incubated at room temperature for one hour. After washing, 50 µl of either Anti-Mouse IgG (γ-chain specific), Anti-mouse IgG1, Anti-mouse IgG2a (Sigma–Aldrich, USA) or Anti-monkey IgG in 1:5000 dilutions were added to each well and plates were incubated at room temperature for one hour. After 4 rounds of wash, 100 µl of Tetramethylbenzidine (TMB) (RaziBIOTech) were added to each well, followed by 10 min of incubation. Finally, 1 N HCl was used as a stop reagent and the absorbance was measured by BioTek® 800™ TS at 450 nm and 630 nm. To calculate endpoint titers, the baseline serum samples of 30 mice, were used and calculated as described by Frey et al.44 (link).
5
Serum IgG and Cytokine Response to Ovalbumin
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Serum IgG response against the Ovalbumin protein was examined using ELISA. Ovalbumin (Sigma) was dissolved in coating buffer at 10 μg/ml (Biolegend) and coated on a 96-well plate overnight. The plate was then washed with PBS with 0.2% Tween 20 (PBS-T) and blocked with PBS-T with 10% bovine serum albumin for 1 h. Serum samples were diluted with PBS-T and added to the plates for 2 h. The plate was washed to remove unbound serum components. A secondary antibody (anti-IgG-HRP, Thermo) was added to the plate and incubated for 1 h. The unbound secondary antibody was removed, and the HRP substrate solution (Biolegend) was added to the plate. After a 20-min incubation, absorbance was measured at 570 nm using a plate reader (Tecan, USA).
To measure antigen-specific cytokine response, an ex vivo splenocyte assay was performed. Splenocytes were collected and prepared as a single-cell suspension from animals dosed using oral gavage needles. Cells were seeded at 2.5 × 106/ml in a 24-well plate. Either PBS or SIINFEKL peptide (10 μg/ml, invivogen) were added to the wells and incubated for 48 h. Cell supernatant was collected, and IFN-g was measured using ELISA (Biolegend).
To measure antigen-specific cytokine response, an ex vivo splenocyte assay was performed. Splenocytes were collected and prepared as a single-cell suspension from animals dosed using oral gavage needles. Cells were seeded at 2.5 × 106/ml in a 24-well plate. Either PBS or SIINFEKL peptide (10 μg/ml, invivogen) were added to the wells and incubated for 48 h. Cell supernatant was collected, and IFN-g was measured using ELISA (Biolegend).
6
Serum antibody measurement in mice
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Fifty µl of blood was collected from the facial veins of mice on day - 1, week 7 and 12. The serum was taken after centrifugation at 6000 g for 10 minutes and frozen at -80. The samples were thawed and diluted 1000 times and used for ELISA. The protocol by Yilmaz et al. was followed (Yilmaz et al., 2021) (link).
Brie y, AChR protein was diluted in coating buffer (BioLegend, cat#421701) and the 96-well ELISA plate was incubated overnight at 4°C. The excess protein was washed with Wash Buffer (PBS 0.05% Tween (Sigma, cat#9005-64-5)). The plates were blocked with 5% FBS (HyClone, cat#SH30071.03IR25-40) in PBS for 2 h and afterwards washed 3 times with Wash Buffer. The serum was added and incubated for 2 hours at room temperature. After 3 washes with Wash buffer anti-IgG1 (Southern Biotech, cat#1071-05), IgG2b (Southern Biotech, cat#1091-05), IgG3 (Southern Biotech, cat#1101-05) and IgM (Southern Biotech, cat#1021-05) were added with 5000-fold dilution. The ELISA experiments were performed two times.
Brie y, AChR protein was diluted in coating buffer (BioLegend, cat#421701) and the 96-well ELISA plate was incubated overnight at 4°C. The excess protein was washed with Wash Buffer (PBS 0.05% Tween (Sigma, cat#9005-64-5)). The plates were blocked with 5% FBS (HyClone, cat#SH30071.03IR25-40) in PBS for 2 h and afterwards washed 3 times with Wash Buffer. The serum was added and incubated for 2 hours at room temperature. After 3 washes with Wash buffer anti-IgG1 (Southern Biotech, cat#1071-05), IgG2b (Southern Biotech, cat#1091-05), IgG3 (Southern Biotech, cat#1101-05) and IgM (Southern Biotech, cat#1021-05) were added with 5000-fold dilution. The ELISA experiments were performed two times.
7
CAR+ T Cell Stimulation Assay
8
CAR+ T Cell Stimulation Assay
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1×105 CAR+ T cells and 1×105 tumor cells were cultured in 200uL CM in 96-well flat bottom plates for 24 hours. For idiotype stimulation, serial dilutions of 1A7 were crosslinked in 1X Coating Buffer (BioLegend) overnight at 4C on Nunc Maxisorp 96-well ELISA plates (Thermo Scientific). Wells were washed once with PBS and 1×105 CAR+ T cells were plated in 200uL CM and cultured for 24h. Triplicate wells were plated for each condition. Culture supernatants were collected and analyzed for IFNγ and IL-2 by ELISA (BioLegend).
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