Briefly, plates (Corning; catalog no. 3690) were coated with 100 ng/well HBsAg (North China Pharmaceuticals Co., Ltd.) in phosphate-buffered saline (PBS) at 4°C overnight and blocked with 3% milk (wt/vol) diluted in PBS at 37°C for 1 h. Cell supernatant or plasma was serially diluted in 1% bovine serum albumin (BSA) buffer. Plates were washed 3 times in PBS-T (0.05% Tween 20 in PBS) and then incubated with diluted samples at 37°C for 1.5 h. Then, plates were incubated with secondary antibody, horseradish peroxidase (HRP)-conjugated anti-human IgG Fc antibody (Thermo Fisher) for IgG antibody and Fc recombinant protein, and mouse anti-His antibody (GenScript) at 37°C for scFv antibody at 37°C for 45 min after washing for 5 times in PBS-T. Plates were washed 5 times and then incubated with ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] at 37°C for 15 min. Analysis was quickly conducted at wavelength of 405 nm by a microplate reader (BioTek).
Hrp conjugated anti human igg fc antibody
Manufactured by Thermo Fisher Scientific
The HRP-conjugated anti-human IgG Fc antibody is a laboratory reagent used for the detection of human immunoglobulin G (IgG) in biological samples. It is a secondary antibody that is conjugated with the enzyme horseradish peroxidase (HRP), which can be used in various immunoassay techniques to amplify and visualize the target IgG signal.
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Lab products found in correlation
1 step ultra tmb, by Thermo Fisher Scientific (2 mentions)
Spectramax paradigm plate reader, by Molecular Devices (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Mouse anti his antibody, by GenScript (1 mentions)
Microplate spectrophotometer, by Agilent Technologies (1 mentions)
Monoclonal anti flag hrp antibody, by Merck Group (1 mentions)
5 protocols using hrp conjugated anti human igg fc antibody
1
ELISA for Quantifying HBsAg Antibodies
2
Antibody Binding Assay Protocol
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Costar half-area high binding assay plates (Corning #3690) were coated with purified protein at 100 ng/well in PBS overnight at 4°C, and blocked with PBS buffer containing 3% milk powder (w/v) at 37°C for 1 h. Serially diluted antibody solutions were added. After reacting for 1.5 h at 37°C, the plate was washed 3 times with PBST (0.05% Tween 20 in PBS). For polyclonal phage ELISA, phages from each round of panning were incubated with immobilized antigen and bound phages were detected with anti-M13- HRP polyclonal antibody (Pharmacia). For the purified antibody binding assay, serially diluted antibody solutions were added and incubated for 1.5 h at 37°C. The bound antibodies were detected with monoclonal anti-FLAG-HRP antibody (Sigma-Aldrich) for UdAb and HRP-conjugated anti-human IgG Fc antibody (ThermoFisher) for IgG antibody. The enzyme activity was measured with the subsequent addition of substrate ABTS and signal reading was carried out at 405 nm using a Microplate Spectrophotometer (Biotek). The EC50 was extrapolated by fitting the data to a sigmoidal dose-response curve with variable slope, using GraphPad Prism version 8.0.
3
ELISA Binding Assay for HSV-Specific mAbs
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The ability for the HSV-specific mAbs to bind to gD was evaluated via an ELISA. Briefly, the wells of a high-binding 96 well plate were coated with 1 μg/mL gD in sodium bicarbonate buffer pH 9.4 and incubated overnight at 4°C. The plates were washed 5x with 1x PBS, 0.1% BSA, 0.05% Tween-20 and blocked with 1x PBS with 2.5% BSA overnight at 4°C. The plates were washed 5x. Antibodies were serially diluted in 1x PBS with 0.1% BSA over a seven point two-fold dilution curve (10.66 nM – 0.16 nM), added to the plates, and incubated at room temperature for 1 hour. The wells were washed 5x and incubated with 100 μl/well with an HRP-conjugated anti-human IgG Fc antibody (1:10000 dilution, Invitrogen) for 1 hour. Wells were washed a final time before being incubated with 100 μL/well 1-step Ultra TMB (Invitrogen) for 5 minutes. The reaction was halted with 100 μL/well 1N H2SO4. The plate was read at 450 nm on a SpectraMax Paradigm Plate Reader (Molecular Devices). Buffer only wells were used as a control and the assay was performed in technical replicate.
4
Quantifying Antibody Binding Specificity
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To measure the binding activities of Ab10 to mutated Gn, Ab10 scFv-Cκ antibody and an anti-influenza virus hemagglutinin antibody (clone 12CA5; Bio X Cell, Lebanon, NH, USA) were coated on a microplate, in parallel. Then, plates were blocked with 3% skim milk in PBS for 1 h at room temperature. Transiently transfected cell supernatant containing recombinant Gn-Fc-HA proteins with alanine substitution was added to each well. After incubation for 2 h at room temperature, the microplate was washed three times with 0.05% Tween20 in PBS solution. Then, HRP-conjugated anti-human IgG Fc antibody (31423; Invitrogen) diluted in blocking buffer was added to each well. The plate was 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). Relative reactivity was calculated using absorbance values (Abs) as follows: % relative reactivity = [100 × {(Abs of mutant captured by Ab10) / (Abs of mutant captured by HA antibody)} / {(Abs of wildtype captured by Ab10) / (Abs of wildtype captured by HA antibody)}].
5
Binding Assay for HSV-specific mAbs
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The ability for the HSV-specific mAbs to bind to gD-2 was evaluated via an ELISA. Briefly, the wells of a high-binding 96 well plate were coated with 1 μg/mL gD-2 in sodium bicarbonate buffer pH 9.4 and incubated overnight at 4°C. The plates were washed 5x with 1x PBS, 0.1% BSA, 0.05% Tween 20 and blocked with 1x PBS with 2.5% BSA overnight at 4°C. The plates were washed 5x. Antibodies were serially diluted in 1x PBS with 0.1% BSA over a seven point 2-fold dilution curve (10.66 nM–0.16 nM), added to the plates, and incubated at room temperature for 1 h. The wells were washed 5x and incubated with 100 μL/well with an HRP-conjugated anti-human IgG Fc antibody (1:10000 dilution, Invitrogen) for 1 h. Wells were washed a final time before being incubated with 100 μL/well 1-step Ultra TMB (Invitrogen) for 5 min. The reaction was halted with 100 μL/well 1N H2SO4. The plate was read at 450 nm on a SpectraMax Paradigm Plate Reader (Molecular Devices). Buffer only wells were used as a control and the assay was performed in technical replicate.
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