A competition ELISA was used to determine epitope specificity, i.e., whether sera from vaccinated animals contained Abs recognizing epitopes similar to the well-characterized Env-specific human broadly neutralizing mAb PG9. Dilutions of sera were preincubated for 10 min at room temperature (RT) in wells of plates (Immulon 4HBX) coated with antigen at a concentration of 1 μg/mL, followed by incubation for 2 hours at RT with a biotinylated PG9. The concentration for biotinylated PG9 used was predetermined in competition with its nonbiotinylated IgG. Plates were washed with PBS containing 0.02% Tween 20 before incubation (1 hour at RT) with Pierce high sensitivity streptavidin–horseradish peroxidase (HRP) reporter reagent (product 21130; Thermo Fisher Scientific). The plates were washed with PBST. The bound HRP enzyme activity was revealed by adding TMB (3,3',5,5'-Tetramethylbenzidine) substrate solution (Thermo Fisher Scientific), and the reaction was stopped by adding 2M sulfuric acid. Plates were then read at a wavelength of 450 nm. A reduction in signal reflects the presence of Abs in serum that competed with the labeled mAb PG9 for binding to the plate-bound antigen.
3 3 5 5 tetramethylbenzidine tmb substrate solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution is a colorimetric reagent commonly used in enzyme-linked immunosorbent assays (ELISA) and other immunoassays. It serves as a substrate for the enzyme-catalyzed reaction, resulting in the development of a colored product that can be measured spectrophotometrically.
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Lab products found in correlation
Multiskan go, by Thermo Fisher Scientific (2 mentions)
96 well immunoplate, by Thermo Fisher Scientific (1 mentions)
Superblock blocking buffer, by Thermo Fisher Scientific (1 mentions)
Microtiter plate reader, by Agilent Technologies (1 mentions)
96 deep well plate, by Corning (1 mentions)
96 well high binding microplates, by Corning (1 mentions)
Versamax elisa microplate reader, by Molecular Devices (1 mentions)
Mouse anti his antibody, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Streptavidin hrp, by R&D Systems (1 mentions)
Softmax pro v5, by Molecular Devices (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti gst cryptate, by PerkinElmer (1 mentions)
Streptavidin coated 384 well plates, by Thermo Fisher Scientific (1 mentions)
Streptavidin xl665, by PerkinElmer (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Mncl2, by Merck Group (1 mentions)
8 protocols using 3 3 5 5 tetramethylbenzidine tmb substrate solution
1
Epitope Specificity of Vaccine-Induced Antibodies
2
Quantification of Clostridium difficile Toxin A
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Total TcdA amount was quantified from cytosol and supernatants by enzyme-linked immunosorbent assay (ELISA). Briefly, 1.5 mL of culture was harvested by centrifugation for 4 min at 13,000 rpm. Supernatants were collected and bacterial pellets were frozen at −20 °C. The frozen bacteria were thawed and sonicated. Cytoplasmic fraction were obtained by centrifuging the lysates (3 min at 13,000 rpm). The supernatant and cytosol fractions were then analyzed by ELISA. A 96-well immuno-plate (Nunc Maxisorp) was coated with 2 µg/mL of anti-toxin A rabbit polyclonal antibody (Abcam, Inc.) overnight at 4 °C. The coated wells were washed and incubated with Superblock blocking buffer (Thermo Fisher Scientific) for 1 h. The wells were then washed and air-dried. Samples were added into the wells, and the plate was incubated at 37 °C for 90 min. After washings, 0.2 µg/mL of an anti-toxin A chicken horseradish peroxidase (HRP) antibody (LSBio) was added in each well and the plate was incubated for 1 h at 37 °C. The wells were washed and incubated with a TMB (3,3′,5,5′-tetramethylbenzidine) substrate solution (Thermo Fisher Scientific) for 15 min in the dark. The stop solution (H2SO4; 0.2 M) was added into each well and the absorbance of the reaction was read at 450 nm.
3
SARS-CoV-2 S-ECD Binding Assay
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Individual colonies of the ninety-six clones selected from the fourth round of biopanning were inoculated into 1 mL of SB containing 50 μg/mL of carbenicillin in 96-deep-well plates (Axygen, Union City, CA, USA) and incubated at 37 °C for 5 h. Subsequently, 1010 pfu of VCSM13 phages (Agilent) were added to the plates and incubated overnight at 37 °C. The plates were centrifuged at 2000× g, and the phage supernatant was used for ELISA. Briefly, 96-well high binding microplates (Corning, Corning, NY, USA) were coated with 0.1 μg of SARS-CoV-2 S-ECD (Sino#40589-V08B1) or 3% BSA in PBS and incubated overnight at 4 °C. After blocking using 3% (w/v) BSA in PBS, the plates were incubated with 100 μL of phage supernatant at 37 °C for 2 h. After three rounds of washing with PBS containing 0.05% (v/v) Tween 20 (PBST), horseradish peroxidase (HRP)-conjugated anti-hemagglutinin (HA) antibody (1:3000; Bethyl Laboratories, Montgomery, TX, USA) was added and incubated at 37 °C for 1 h. Colorimetric detection was performed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was terminated by the addition of 1 M H2SO4, and absorbance was measured at 450 nm using a microtiter plate reader (Bio-Tek Instruments, Winooski, VT, USA).
4
IgG1 Antibody Binding Assay
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In order to measure the binding activities of the Ab10 and MAb4-5 IgG1 antibodies, 96-well half-area microplates (3690; Corning, Corning, NY, USA) were coated with Gn-Fc fusion protein and incubated at 4°C overnight. Plates were blocked with 3% skim milk in PBS for 1 h at room temperature. The plates were then washed with PBS and received antibodies that were 10-fold serially diluted from 1 μM to 10 μM in blocking buffer. The plates were then incubated for 2 h at room temperature and washed three times with 0.05% Tween20 in PBS solution. Then, 50 μL of HRP-conjugated anti-human Ig kappa light chain antibody (AP502P; Chemicon, Temecula, CA, USA) diluted in blocking buffer (1:5000) was added into each well. Then, plates were incubated for 1 h at room temperature. After washing, each well received 50 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (34028; Thermo Scientific). The color reaction was stopped by adding 50 μL of 2 M sulfuric acid. The absorbance of each well was measured at 450 nm using a microplate spectrophotometer (Multiskan GO; Thermo Scientific).
5
Enzyme-Linked Immunosorbent Assay (ELISA) for Bacterial and Fungal Antibodies
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Briefly, 96-well plates (Maxisorp, Affimetrix eBioscience) were coated with 5 × 106 heat-killed K. pneumoniae or S. pneumoniae or 100 μg/ml C. albicans in bicarbonate/carbonate buffer (100 mM, pH 9.6), blocked with PBS containing 3% BSA (PBS-BSA) and incubated with mouse serum diluted 1/200 in PBS-BSA. IgGs were detected with peroxidase-goat anti-mouse IgG (H+L) and then 3,3′,5,5′-tetramethylbenzidine (TMB) Substrate Solution (ThermoFisher Scientific). Reactions were stopped using 0.16 M sulfuric acid and absorbance measured at 450 nm using a VersaMax ELISA microplate reader (Molecular devices, Sunnyvale, CA). All washing steps were performed using PBS containing 0.05% (v/v) Tween-20.
6
TLR2 Binding and Interaction Assays
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Ninety-six-well plates were coated with purified proteins (1.0 μg/well) and blocked for 1 h with PBS containing 1% (w/v) bovine serum albumin (PBS-BSA). TLR2 was diluted (0.375 to 6 μg/ml) to incubate with the plates overnight at 4 °C and bound TLR2 was detected by incubating for 1 h with a mouse anti-His antibody (Sigma-Aldrich) and then 30 min with an HRP-conjugated secondary antibody (DAKO, Glostrup, Denmark). TLR2 was also coated (2 μg/ml) to incubate with NCL, NCL-HA, or the NCL peptides. Bound NCL was detected using a rabbit anti-NCL antibody and bound NCL-HA and its mutants were detected using a mouse anti-HA antibody (1 μg/ml), followed by HRP-conjugated secondary antibodies. Plate-coated TLR2 was also pre-incubated with mouse TLR2 or TLR4-blocking antibodies (5 μg/ml) before incubation with NCL or NCL-HA, and bound NCL or NCL-HA was detected using a rabbit anti-NCL antibody. Coated TLR2 was incubated with biotin-Ahx-tagged peptides and the bound peptides were detected with streptavidin-HRP. Plates were all developed using the 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) substrate solution (ThermoFisher Scientific).
7
IgG Detection in Mouse Vaginal Samples
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After preparation of vaginal smears, cervico-vaginal washings were spun at 24,000xg for 10 min at 4°C. Sample collection was performed during each cycle stage every 24 hours so that endogenous mouse IgG, which had accumulated in this discrete time window, could be detected in the samples using an IgG-specific ELISA kit (Mabtech, Nacka Strand, Sweden), as per manufacturer's instructions with some modifications. Briefly, instead of using the supplied alkaline phosphatase (ALP)-conjugated goat anti-mouse IgG detection antibody included in the kit, a biotinylated version of the same detection antibody was used (Mabtech, Nacka Strand, Sweden). As an additional step, streptavidin-HRP (R&D Systems, Minneapolis, USA) was added to the ELISA plates for 30 minutes followed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Scientific, Rockford, USA). Color development was stopped after 10 minutes with 1 N H2SO4 and plates were read at 450 nm with a tunable VersaMax microplate reader in conjunction with the SoftMax Pro v.5.3 software (Molecular Devices, Sunnyvale, USA).
8
Plk1 Kinase Interaction Assay
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(Biotin-DPPLHS-pT-AI), and poloboxtide (MAGPMQS-pT-PLNGAKKK) peptides were obtained from Peptron (Daejeon, South Korea). Full-length Human Plk1 expressed as an N-terminal glutathione S transferase (GST)fusion protein was obtained from Carna (Kobe, Japan). Streptavidin-XL665 and anti-GST-cryptate were purchased from Cisbio (Codolet, France). Full-length Human Plk1 fused with enhanced green fluorescent protein (EGFP) was prepared from mitotic HEK293T lysates expressing EGFP-Plk1. Streptavidin-coated 384-well plates, horseradish peroxidase (HRP)-conjugated secondary antibodies, and a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution were obtained from Thermo Fisher Scientific (Waltham, MA, U.S.A.). To detect bound EGFP-Plk1, an anti-Plk1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Purpurogallin and poloxin were purchased from TCI America (Portland, OR, U.S.A.) and Sigma-Aldrich (St. Louis, MO, U.S.A.), respectively. All other reagents including MgCl 2 , MnCl 2 , and dithiothreitol were obtained from Sigma-Aldrich.
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