Biotin-conjugated antibodies specific to mouse IgE were obtained from BD Bio-Sciences (San Jose, CA, USA). The p-nitrophenyl phosphate compound was sourced from Sigma (St. Louis, MO, USA), and Streptavidin alkaline phosphatase was acquired from Jackson ImmunoResearch (West Grove, PA, USA). Folin reagent was obtained from Bio-Rad (Hercules, CA, USA). Specific reagents, including the IgE Mouse Uncoated ELISA Kit with Plates, StreptAvidin-HRP, TMB substrate, MCPT-1 (mMCP-1) Mouse Uncoated ELISA Kit with Plates, Avidin-HRP, and TMB substrate, were procured from Invitrogen (Waltham, MA, USA). The Tissue Protein Extraction Reagent (T-PERTM), a proprietary detergent with a composition of 25 mM bicine and 150 mM sodium chloride at pH 7.6, was obtained from ThermoFisher Scientific (Waltham, MA, USA). For protease inhibition, a cocktail of serine, cysteine, and acid proteases, along with aminopeptidases, was acquired from Sigma-Aldrich (St. Louis, MO, USA).
Tmb substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
TMB substrate is a chromogenic substrate used in enzyme-linked immunosorbent assay (ELISA) and other immunodetection methods. It is a two-component system that, when combined, produces a blue color upon oxidation by the enzyme horseradish peroxidase (HRP). The intensity of the blue color is proportional to the amount of HRP present, allowing for quantitative analysis of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (11 mentions)
Avidin hrp, by Thermo Fisher Scientific (8 mentions)
96 well high binding polystyrene plate, by Corning (8 mentions)
Tween 20, by Merck Group (7 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (7 mentions)
Elisa plate, by Corning (6 mentions)
Stop solution, by Thermo Fisher Scientific (6 mentions)
High binding elisa plate, by Corning (6 mentions)
Tmb stop solution, by Thermo Fisher Scientific (6 mentions)
Microplate reader, by Agilent Technologies (5 mentions)
Superblock, by Thermo Fisher Scientific (5 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (5 mentions)
96 well half area plate, by Corning (5 mentions)
96 well plate, by Thermo Fisher Scientific (5 mentions)
Skim milk, by Merck Group (5 mentions)
Prism 9, by GraphPad (5 mentions)
Maxisorp plate, by Thermo Fisher Scientific (4 mentions)
Prism 5, by GraphPad (4 mentions)
High binding 96 well plate, by Corning (4 mentions)
Microplate reader, by Thermo Fisher Scientific (4 mentions)
351 protocols using tmb substrate
1
Mouse IgE ELISA Assay Protocol
2
Quantitative TNFR-Fc Titer Measurement
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To determine TNFR-Fc titers, conditioned medium was analyzed by the sandwich enzyme-linked immunosorbent assay method. Essentially, 96-well plates (Constar/Corning, USA) were pre-coated with a goat anti-human IgG primary antibody (CW bio, China) at 37°C overnight. Following a series of wash steps, the wells were blocked with bovine serum albumin in PBS and Tween 20. Bound recombinant antibodies were detected using TMB substrate (Invitrogen, USA) following incubation of each well with a mouse anti-human IgG HRP-conjugated secondary antibody (CW bio, China). Following 30 mins of incubation at room temperature, the TMB substrate reaction was terminated with 2 M H2SO4 prior to reading at 450 nm using a microplate reader (Thermo, USA). Data were collected and analyzed using GraphPad Prism 5. Quantification was based on a dilution series to obtain a standard curve of known TNFR-Fc concentrations.
3
Enzyme-Linked Immunosorbent Assay for Anti-HSF1-PO4 Antibodies
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Phosphorylated and non-phosphorylated synthetic peptides or recombinant HSF1-PO4 protein (Abcam #115508) were diluted in carbonate/bicarbonate coating buffer (4 µg/mL) and incubated overnight in 96-well NUNC maxisorp plates at 4 °C. Plates were washed with wash buffer (PBS/0.05% v/v Tween-20) and blocked with PBS/1% BSA for a minimum of 1 h at 37 °C. After washing, plasma/sera were added (duplicate wells) and incubated at 4 °C overnight (1:10 or 1:20 dilution in wash buffer). After washing, HRP-conjugated goat anti-human IgA alpha chain (1:10,000, Abcam #98558) was added and incubated at 37 °C for 1.5 h. Following washing, the reaction was developed using TMB substrate (Invitrogen, Waltham, MA, USA) and stopped with 1 M HCl. Absorbance was immediately read at 450 nm (Optical Density (OD)450nm) using a Multiskan Go plate reader (Thermo Scientific, Waltham, MA, USA). Where specified, OD450nm values were normalised against values from a single patient sample consistently run across multiple experiments to account for inter-assay variability. The selected sample (OV0023, refer to Supplementary Table S2 ) had a mid-range anti-HSF1-PO4 or anti-peptide response (ranging OD450nm 0.41–1.0, depending on the target) and was thus chosen to avoid extreme measurements that could introduce bias.
4
Binding Assay for Antibody-Antigen Interactions
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The mAbs were tested for binding against 16055 NFL TD CC trimer, gp120, and the gp120 V2b(180–194) deletion mutant with residues replaced with GAG (Martinez-Murillo et al., 2017 (link)). MaxiSorp 96-well plates (Nalgene Nunc International) were coated at 2 µg/ml with WT gp120, gp120 V2b, or with an anti-His tag mAb (AD1.1.10; R&D Systems) to capture the His-tagged trimer in PBS at 4°C overnight. After incubation with blocking buffer (PBS containing 2% nonfat milk), the mAbs were added and incubated for 1 h at 37°C. Binding was detected by secondary HRP-conjugated anti-human Fcγ Ab (Jackson ImmunoResearch) at 1:10,000 for 1 h. The signal was developed by addition of TMB substrate (Invitrogen) for 5 min, reactions were terminated with 1 N sulfuric acid, and the OD was read at 450 nm. Between each incubation step, the plates were washed six times with PBS containing 0.05% Tween-20.
5
Quantifying ACE2 and SARS-CoV-2 Spike Binding
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The analysis of ACE2 concentration in culture supernatants was performed according to the manual of ELISA Kits (ab235649, abcam). Briefly, EVs were added to the plate followed by the addition of anti‐hACE2 antibody cocktails which contained capture antibody and detector antibody. The plate was incubated for 1 h at RT on a plate shaker at 450 rpm. After washing steps, TMB substrate was added and incubated for 15 min. The chromogenic reaction was quantified following the addition of stop solution. The absorbance of the samples was measured at 450 nm.
The binding capacity of EV‐ACE2 against spike glycoprotein (RBD) was measured by SARS‐CoV‐2 surrogate virus neutralization test kit (GenScript, China).[77 ] Briefly, EVs were pre‐incubated with the HRP‐RBD to allow the binding of EVs to HRP‐RBD. Then the mixture was added to the plate pre‐coated with human ACE2 protein. After washing steps, TMB substrate (Invitrogen) was added to each well and the plate was incubated in the dark at ≈20–25 °C for 15 min. The chromogenic reaction was quantified following the addition of stop solution (KPL SeraCare). The absorbance of the samples was measured at 450 nm. All experiments were performed according to the manufacturer's instructions.
The binding capacity of EV‐ACE2 against spike glycoprotein (RBD) was measured by SARS‐CoV‐2 surrogate virus neutralization test kit (GenScript, China).[
6
SARS-CoV-2 Spike Protein Antibody Assay
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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).
7
Sandwich ELISA for Dimeric TFF3 Detection
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A sandwich ELISA was established based on our developed ELISA using the FITC and anti-FITC detection system [47 (link)]. The ELISA plate (Costar) was coated with purified anti-TFF3 mAbs (1 μg/ml) in carbonate/bicarbonate coating buffer of pH 9.6 at 4 °C overnight. After washing, the plate was blocked with 2% skimmed milk in PBS at 37 °C for 1 h. Various concentrations of the dimeric TFF3 or the saliva samples were then added and the plates were incubated for 1 h. FITC-labeled anti-TFF3 mAbs (1 μg/ml) were added and the plates were incubated at 37 °C for 1 h. HRP-conjugated sheep anti-FITC antibodies (Thermo Scientific) were used to detect the bound antibodies at 37 °C for 1 h. Thereafter, TMB substrate (Invitrogen) was added. The reaction was stopped using 1 N HCl and the absorbance was measured at OD450 nm.
8
RBD-DP Binding Assay with ACE2-Fc
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High binding 96-well plates (Corning) were coated with 100 μL of 2 μg/mL RBD-DP diluted in 0.1 M Sodium Carbonate/Bicarbonate buffer overnight in duplicates at 4 °C. The next day, the wells were blocked with 1% (w/v) BSA in PBS, 0.05% Tween (PBS-T). After four washes with PBS-T, 100 μL serially diluted ACE2-Fc (in-house, 9.75–20,000 ng/μl) was added to the wells. The plates were incubated at room RT for 2 h to allow ACE2-Fc to bind to RBD-DP. Plates were washed four times with PBS-T followed by the addition of 100 μL 1:2000 diluted HRP-conjugated anti-human IgG antibody (Life technologies) and incubated for 1 h at RT. After this binding step, the plates were washed four times with PBS-T and two times with ultra-pure water. Finally, 100 μL TMB substrate (Invitrogen) was added and incubated for 15 min in the dark. The reaction was terminated with 100 μL stop solution (Invitrogen) and the absorption was measured at 450 nm using a Synergy H1 microplate reader (BioTek). Binding signals were reported as the background subtracted signal divided by the background. Hill equation was used to fit RBD-DP:ACE2-Fc binding curve and apparent KD was calculated based on this fit (GraphPad Software).
9
SARS-CoV-2 RBD Protein ELISA
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96-well Maxisorp plates (Nunc) were coated with 2 µg/ml of SARS-CoV-2 RBD protein (Genscript) in bicarbonate buffer overnight at 4°C. Wells were blocked using BD OptEIA assay diluent (BD) for 1 h at 37°C. Heat-inactivated sera were depleted for serum IgG using Gullsorb™ Human IgG Inactivation Reagent (Meridian Bioscience) as per manufacturer’s recommendations. Depleted and undepleted sera were then further diluted to a final concentration of 1:200, added into ELISA microwells and incubated for 1 h at 37°C. Following extensive washing, anti-human IgM-HRP (Life Technologies) or anti-human IgG-HRP (Santa Cruz) diluted 1:10,000 was added and incubated for 30 min at 37°C. The chromogenic reaction was quantified following the addition of TMB substrate (Invitrogen) and stop solution (KPL SeraCare). The absorbance of the samples was measured at 450 nm and the background at 570 nm. The results are presented as the OD difference of 450 and 570 nm.
10
Construction and Characterization of Anti-PD-L1-OX40 Bispecific Antibody
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Example 2
Anti-PD-L1-OX40 bispecific antibody was constructed as shown in the
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