96-well microtiter plates were coated with 100 µL 10 µg/mL recombinant FRα in 0.05 M sodium bicarbonate (pH 9.6) and the plate was kept overnight at 4 °C. After washing the plate three times with TBST, the plate was blocked with 3% BSA at 37 °C for 2 h. For polyclonal phage ELISA, after each round of screening, the phage eluate was amplified and 100 μL 1010 phages diluents were added to each well and incubated for 2 h. To analysis individual phage clones, each phage clone was amplified and 1010 phages were added to each well (100 µL /well) and incubated for 2 hours at 37 °C. After washing the plate for 3 times with TBST and 3 times with TBS, 100 µL of HRP-conjugated anti-M13 antibody (1:10000, Sino Biological, Inc., Beijing) was added and the plate was incubated for 1 hour at 37 °C. After washing, the tetramethylbenzidine (TMB) (Sangon Biotech Co., Ltd., Shanghai) substrate (100 μL/well) was added to the wells and the reaction lasted for 15 minutes. The reactions were stopped with 2 M sulfuric acid (50 μL/well). The absorbance of each well at 450 nm was detected with an automated ELISA reader.
Hrp conjugated anti m13 antibody
Manufactured by Sino Biological
Sourced in China, United States
The HRP-conjugated anti-M13 antibody is a laboratory reagent that can be used to detect and quantify the presence of the M13 bacteriophage in various experimental systems. It is a highly specific antibody that has been conjugated with the enzyme horseradish peroxidase (HRP), which allows for easy detection and measurement of the target protein using standard colorimetric or chemiluminescent assays.
Automatically generated - may contain errors
Lab products found in correlation
Ap conjugated donkey anti human igg, by Jackson ImmunoResearch (3 mentions)
Pnpp substrate, by Merck Group (3 mentions)
Tmb substrate, by Merck Group (3 mentions)
Tetramethylbenzidine tmb, by Sangon (2 mentions)
Tmb substrate solution, by Merck Group (1 mentions)
96 well half area microplate, by Corning (1 mentions)
Spectramax 190 microplate reader, by Molecular Devices (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Promega (1 mentions)
Maxisorp microtiter plate, by Thermo Fisher Scientific (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Fluostar omega reader, by BMG Labtech (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Tetramethylbenzidine, by Solarbio (1 mentions)
10 protocols using hrp conjugated anti m13 antibody
1
Phage Display Screening for FRα Binders
2
SARS-CoV-2 RBD Phage ELISA Assay
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A polyclonal phage ELISA was performed using pools of purified phage from each library stock. A 96-Well Half-Area Microplate (Corning, Cat. 3690, New York, NY, USA) was coated overnight at 4 °C, with 30 μL per well of 1 μg/mL SARS-2 RBD (Sino Biological, Cat. 40592-V08H, Beijing, China), and each well was blocked with 5% skimmed milk in PBS (MPBS) for 1 h at room temperature. Phage pools (~1012 phage particles) were also blocked in MPBS for 1 h at room temperature and then blocked phage pools were added to the SARS-2 RBD-coated plate and incubated for 1 h at 37 °C. After washing four times with PBST, the horseradish peroxidase (HRP)-conjugated anti-M13 antibody (1:5000, Sino Biological, Cat. 11973-MM05, Beijing, China) was incubated for 1 h at 37 °C. After washing four times with PBST, a TMB substrate solution (Sigma-Aldrich, Cat. T0440, St. Louis, MO, USA) was added for 8 min, and the reaction was stopped with 1 N sulfuric acid (Merck, Cat. 100731, Darmstadt, Germany). The absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnydale, CA, USA).
3
Evaluating scFv-Phage Binding to SARS-CoV-2 S1
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The binding capacity of scFv-phage suspensions to SARS-CoV-2 S1 protein were detected by phage ELISA. In detail, the bacteria solution (50 μL) from each antibody phage library was inoculated into 2YT medium (5 mL) and cultured at 37 °C for 3 h with shaking and transferred into of 2YT-AG medium (5 mL; containing 100 μg/mL ampicillin, 2% glucose) and M13KO7 phages were added. The above 2YT-AG medium was incubated at 37 °C for 2.5 h at 150 rpm in the shaker and then centrifuged at 1000g for 15 min at 4 °C. The precipitate was resuspended in 2YT-AK (400 μL) and cultured at 37 °C overnight with shaking at 250 rpm on the shaker. The bacterial culture solution was centrifuged and the supernatant was collected for phage ELISA analysis.
The wells of 96-well Maxisorp microtiter plate (Nunc, Roskilde, Denmark) were coated with S1 protein in carbonate buffer solution (CBS, 100 μL) overnight at 4 °C and then blocked with bovine serum albumin (BSA, 300 μL) for 2 h at 37 °C. The scFv-phage was added to each well for 2 h incubation at 37 °C. The bound scFv-phage was detected with horseradish peroxidase (HRP)-conjugated anti-M13 antibody (Sino Biological, Beijing, China). The color was developed using TMB (Promega Biotech, Beijing, China) for 10 min and the absorbance was read at 450 nm.
The wells of 96-well Maxisorp microtiter plate (Nunc, Roskilde, Denmark) were coated with S1 protein in carbonate buffer solution (CBS, 100 μL) overnight at 4 °C and then blocked with bovine serum albumin (BSA, 300 μL) for 2 h at 37 °C. The scFv-phage was added to each well for 2 h incubation at 37 °C. The bound scFv-phage was detected with horseradish peroxidase (HRP)-conjugated anti-M13 antibody (Sino Biological, Beijing, China). The color was developed using TMB (Promega Biotech, Beijing, China) for 10 min and the absorbance was read at 450 nm.
4
Phage ELISA for RBD Binders
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A total of 106 individual clones obtained from the third round of biopanning were measured using a phage ELISA. Each clone was inoculated into 1 mL of 2YT-GA and grown until the OD600 reached 0.5. After being infected with the M13KO7 helper phage, the bacterial cultures were incubated overnight at 30 °C. Following centrifugation, 500 μL of phage supernatants was collected for subsequent phage ELISA.
The wells of a 96-well microplate (Costar, Corning, NY, USA) were coated with 50 ng of WT RBD, while the negative control consisted of 5% BSA. Following an overnight incubation at 4 °C, a blocking solution of 300 μL/well was added to the microplate for blocking. Subsequently, 100 μL of each phage supernatant was added to the wells containing RBD and BSA. After incubation at 37 °C for one hour, the microplate was washed three times with PBST to remove nonbinding phages. The bound phages were detected by incubating them with HRP-conjugated anti-M13 antibody (1:10,000, Sino Biological) at 37 °C for one hour. Then, TMB substrate and stop solution (Solarbio) were sequentially added. The absorbance at 450 nm was measured using a SpectraMax i3x plate reader (Molecular Devices, San Jose, CA, USA). The RBD and BSA well reads were recorded, and the binding ratio was calculated.
The wells of a 96-well microplate (Costar, Corning, NY, USA) were coated with 50 ng of WT RBD, while the negative control consisted of 5% BSA. Following an overnight incubation at 4 °C, a blocking solution of 300 μL/well was added to the microplate for blocking. Subsequently, 100 μL of each phage supernatant was added to the wells containing RBD and BSA. After incubation at 37 °C for one hour, the microplate was washed three times with PBST to remove nonbinding phages. The bound phages were detected by incubating them with HRP-conjugated anti-M13 antibody (1:10,000, Sino Biological) at 37 °C for one hour. Then, TMB substrate and stop solution (Solarbio) were sequentially added. The absorbance at 450 nm was measured using a SpectraMax i3x plate reader (Molecular Devices, San Jose, CA, USA). The RBD and BSA well reads were recorded, and the binding ratio was calculated.
5
Phage Display Binding Assay
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Recombinant CD4 (0,5 µg/well) was immobilized on a 96-well Maxisorp plate (Nunc, Thermo Fisher) at 4 °C overnight followed by addition of 100 µL of blocking buffer (5% BSA dissolved in PBS) at 37 °C for 3 h. After two washes with 250 µL of PBST (0.1% tween 20), pre-blocked (5% BSA, 37 °C, 1 h) phages (1010 pfu/well) were added and incubated for 0.5 h with shaking. Unbound phages were removed by 5 consecutive washes and bound phages were incubated with 100 µL of HRP-conjugated anti-M13 antibody (Sino Biological, Beijing, China). Samples were incubated with 100 µL of 3,3′,5,5′-tetramethylbenzidine (Merck, Darmstadt, Germany) in the dark until color development (15 min) and the reaction was terminated with 100 µL of 2M H2SO4. The absorbance was measured on a FLUOstar® Omega reader (BMG Labtech, Ortenberg, Germany) at 450 nm and 540 nm (as reference wavelength).
6
Phage Display Screening for SKOV3 Targets
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After the fourth round of biopanning, 54 phage clones were randomly picked out from titered phage plaques for ELISA. SKOV3 and HEK293 cells were seeded into 96-well plates (4 × 104 cells/well) overnight and then fixed with 4% paraformaldehyde for 20 min at room temperature. 3% H2O2 (100 μl/well) was added, and the plates were placed at room temperature for 30 min to inhibit the activity of endogenous peroxidase. Then cells were blocked with blocking buffer (TBST containing 3% BSA, 250 μl/well) at 37 °C for 2 h. The selected phages (1 × 1010 pfu/well) were added to SKOV3 and HEK293 cells and incubated at 37 °C for 1.5 h. After that, the cells were washed three times with TBST and cultured with HRP-conjugated anti-M13 antibody (diluted at 1:10,000 in 3% BSA, Sino Biological, Beijing, China) for 1 h. Tetramethylbenzidine (100 μl/well, Solarbio Technology Co., Ltd.) were added to the cells and incubated at 37 °C in the dark for 20 min. The incubation was stopped by adding 100 μl/well stop solution. Finally, the 96-well plates were measured at 450 nm using an ELISA reader (Bio-Tek ELX800, USA). Irrelevant phage clone (IRP, an amplified phage randomly selected from the original phage peptide library) and PBS were used as control groups. The relative binding abilities were calculated by ODSKOV3/ODHEK293, the ratio of absorbance over 2.1 was identified as positive clone.
7
Screening Phage Display Library for Target Protein
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96-well polystyrene microtiter plates were coated with a solution of target protein (5 μg/mL) in 0.05 M sodium bicarbonate (pH 9.6) and the plate was kept overnight at 4 °C. After washing the plate three times, the plate was blocked with 0.1 M PBS (pH 7.4) containing 3% NFM at 37 °C for 2 hours (200 μL/well). Individual phage clones were amplified and 1010 phages were added to each well (100 μL/well) and incubated for 2 hours at 37 °C. After washing the plate for 3 times with TBST and 3 times with TBS, 100 μL of HRP-conjugated anti-M13 antibody (1:5000, Sino Biological, Inc., Beijing) was added and the plates were incubated for 2 hours at 37 °C. After washing three times, the tetramethylbenzidine (TMB) (Sangon Biotech Co., Ltd., Shanghai) substrate (100 μL/well) was added and the reaction lasted for 15 minutes. The reactions were stopped with 2 M sulfuric acid (50 μL/well). The absorbance of each well at 450 nm was detected with an automated ELISA reader. Phage clones with high absorbance were selected for sequencing.
8
Rapid ELISA Protocol for Viral Antibody Detection
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Direct ELISA (65 (link)) consisted of coating microtiter plates with 1 × 107 PFU/mL of inactivated VACV IHD-J or ΔH3 or with 2 µg/mL recombinant proteins. For phage ELISA, HRP-conjugated anti-M13 antibody (Sino Biological, USA, Cat# 11973-MM05T-H lot HO13AU601; used at 250 ng/mL) was used following detection with TMB substrate (Millipore, USA). ELISA of both serum and recombinant antibodies was applied with AP-conjugated donkey anti-human IgG (Jackson ImmunoResearch, USA, Cat# 709-055-149 lot 130049; used at 1:2,000 working dilution) following detection using PNPP substrate (Sigma, Israel).
9
Rapid ELISA Protocol for Viral Antibody Detection
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Direct ELISA (65 (link)) consisted of coating microtiter plates with 1 × 107 PFU/mL of inactivated VACV IHD-J or ΔH3 or with 2 µg/mL recombinant proteins. For phage ELISA, HRP-conjugated anti-M13 antibody (Sino Biological, USA, Cat# 11973-MM05T-H lot HO13AU601; used at 250 ng/mL) was used following detection with TMB substrate (Millipore, USA). ELISA of both serum and recombinant antibodies was applied with AP-conjugated donkey anti-human IgG (Jackson ImmunoResearch, USA, Cat# 709-055-149 lot 130049; used at 1:2,000 working dilution) following detection using PNPP substrate (Sigma, Israel).
10
SARS-CoV-2 Spike Protein ELISA Assay
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Direct ELISA33 consisted of coating microtiter plates with 2 μg/ml of recombinant SARS-CoV-2 spike, S1 domain, RBD or NTD subunits. For phage ELISA, HRP-conjugated anti-M13 antibody (Sino Biological, USA, Cat# 11973-MM05T-H lot HO13AU601; used at 1:5000 working dilution) was used following detection with TMB substrate (Millipore, USA). ELISA of both sera and recombinant scFv-Fc human antibodies was applied with AP-conjugated Donkey anti-human IgG (Jackson ImmunoResearch, USA, Cat# 709-055-149 lot 130049; used at 1:2000 working dilution) following detection using PNPP substrate (Sigma, Israel).
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