Maxisorp plates (Nunc) were coated overnight at 4 °C with 5 µg of G, N (NiV Malaysia, Native Antigen Company) or F protein per plate in Carb/Bicarb binding buffer (4.41 g KHCO3 and 0.75 g Na2CO3 in 1 L distilled water). After blocking with 5% milk in PBS with 0.01% tween (PBST), serum in 5% milk in PBST was incubated at RT for 1 hr. Antibodies were detected using affinity-purified antibody peroxidase-labeled goat-anti-monkey IgG (Seracare) in 5% milk in PBST and TMB 2-component peroxidase substrate (Seracare) and read at 450 nm. All wells were washed 3x with PBST in between steps.
Peroxidase labeled goat anti monkey igg
Manufactured by LGC
Sourced in United States
Peroxidase-labeled goat-anti-monkey IgG is a primary antibody conjugate used for the detection and quantification of monkey immunoglobulin G (IgG) in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (14 mentions)
Maxisorp plate, by Thermo Fisher Scientific (9 mentions)
Tmb 2 component peroxidase substrate, by LGC (7 mentions)
Stop solution, by LGC (6 mentions)
Casein, by Thermo Fisher Scientific (6 mentions)
Casein, by LGC (2 mentions)
Expicho, by Thermo Fisher Scientific (1 mentions)
9 protocols using peroxidase labeled goat anti monkey igg
1
SARS-CoV-2 Antibody Detection ELISA
2
SARS-CoV-2 Spike Protein ELISA Assay
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A plasmid encoding the prefusion stabilized SARS-CoV-2 spike protein with a T4 fibritin trimerization motif was obtained from the Vaccine Research Centre, Bethesda, MD, USA and expressed in-house. Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with 100 ng/well spike protein in PBS. Plates were blocked with 100 µL of casein in PBS (Thermo Fisher, Rockville, MD, USA) for 1 h at RT. Serum serially diluted 2× in casein in PBS was incubated at RT for 1 h. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-monkey IgG (Seracare, Milford, MA, USA, 074-11-021) in casein and TMB 2-component peroxidase substrate (Seracare, Milford, MA, USA, 5120-0047), developed for 5–10 min, and reaction was stopped using stop solution (Seracare, Milford, MA, USA, 5150-0021) and read at 450 nm. All wells were washed 3× with PBST 0.1% Tween between steps. Threshold for positivity was set at 3× OD value of negative control (serum obtained from non-human primates prior to start of the experiment) or 0.2, whichever was higher.
3
Recombinant MERS-CoV S Protein Antibody Assay
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A soluble, trimeric recombinant S protein of MERS-CoV (isolate Ca/Jeddah/D42/2014) incorporating amino acids 1 to 1273 and a C-terminal trimerization domain was produced in Chinese hamster ovary cells (expiCHO; Thermo Fisher Scientific) and purified by immunoaffinity chromatography. MaxiSorp plates (Nunc) were coated overnight at room temperature with 5 μg of S protein per plate in PBS. Plates were blocked with 100 μl of casein (Thermo Fisher Scientific) for 90 min at room temperature. Serum (2× serial dilution in casein starting at 100× dilution) was incubated at room temperature for 2 hours. Antibodies were detected using affinity-purified polyclonal antibody peroxidase–labeled goat anti-monkey IgG (SeraCare, 074-11-021) in casein and 3,3′,5,5′-Tetramethylbenzidine (TMB) two-component peroxidase substrate (SeraCare) and read at 450 nm. All wells were washed three times with PBST (PBS-Tween 0.05%) in between steps. Threshold for positivity was set at 3× optical density (OD) value of negative control (serum obtained from NHPs before the start of the experiment).
4
Quantifying SARS-CoV-2 Spike Protein Antibodies
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A plasmid encoding the prefusion stabilized SARS-CoV-2 spike protein with a T4 fibritin trimerization motif was obtained from the Vaccine Research Centre, Bethesda, USA and expressed in-house. Maxisorp plates (Nunc) were coated overnight at 4 °C with 100 ng/well spike protein in PBS. Plates were blocked with 100 μl of casein in PBS (Thermo Fisher) for 1hr at RT. Serum serially diluted 2x in casein in PBS was incubated at RT for 1hr. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-monkey IgG (Seracare, 074-11-021) in casein and TMB 2-component peroxidase substrate (Seracare, 5120–0047), developed for 5–10 min, and reaction was stopped using stop solution (Seracare, 5150–0021) and read at 450 nm. All wells were washed 3x with PBST 0.1% tween in between steps. Threshold for positivity was set at 3x OD value of negative control (serum obtained from non-human primates prior to start of the experiment) or 0.2, whichever one was higher.
5
ELISA for SARS-CoV-2 Antibody Detection
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Enzyme-linked immunosorbent assays (ELISAs) were performed as described previously (8 (link)). Briefly, maxisorp plates (Nunc) were coated overnight at 4°C with 100 ng per well S or RBD protein in PBS. Plates were blocked with 100 μl of casein in PBS (Thermo Fisher Scientific) for 1 hour at room temperature. Serum serially diluted two times in casein in PBS was incubated at room temperature for 1 hour. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat–anti-monkey IgG (SeraCare, 074-11-021) in casein followed by 3,3′,5,5′-tetramethylbenzidine 2–component peroxidase substrate (SeraCare, 5120-0047). The reaction was stopped using stop solution (SeraCare, 5150-0021) and read at 450 nm. All wells were washed four times with PBS with 0.1% Tween 20 in between steps. Threshold for positivity was set at three times the optical density value of negative control (serum obtained from NHPs before start of the experiment) or 0.2, whichever was higher.
6
SARS-CoV-2 Spike Protein IgG and IgM Detection
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Prefusion stabilized SARS-CoV-2 spike protein with a T4 fibritin trimerization motif20 was obtained from the Vaccine Research Centre, Bethesda, USA. Maxisorp plates (Nunc) were coated overnight at 4°C with 100 or 250 ng/well spike protein in PBS for IgG and IgM detection, respectively. Plates were blocked with 100 µl of casein in PBS (Thermo Fisher) for 1hr at RT. Serum serially diluted 2x in casein in PBS was incubated at RT for 1hr or 37°C for 2hr. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-monkey IgG (Seracare, 074–11-021) in casein and TMB 2-component peroxidase substrate (Seracare, 5120–0047), developed for 5–10 min, and reaction was stopped using stop solution (Seracare, 5150–0021) and read at 450 nm or anti-monkey IgM – AP labelled after adding the pNPP substrate and measuring OD values at 405 nm. All wells were washed 4x with PBST 0.1% tween in between steps. Threshold for positivity was set at 3x OD value of negative control (serum obtained from non-human primates prior to start of the experiment) or 0.2, whichever one was higher, or OD of three times the background for the calculation of the IgM endpoint values.
7
ELISA for Evaluating Anti-SARS-CoV-2 Antibodies
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ELISA was performed as described previously26 (link). Briefly, maxisorp plates (Nunc) were coated overnight at 4°C with 50 ng/well S or RBD protein in PBS. Plates were blocked with 100 μl of casein in PBS (Thermo Fisher) for 1hr at RT. Serum diluted 1:6,400 was further 2-fold serially diluted in casein in PBS was incubated at RT for 1hr. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-monkey IgG (Seracare, 074-11-021) in casein followed by TMB 2-component peroxidase substrate (Seracare, 5120–0047). The reaction was stopped using stop solution (Seracare, 5150–0021) and read at 450 nm. All wells were washed 4x with PBST 0.1% tween in between steps. Threshold for positivity was set at 2x OD value of negative control (serum obtained from unvaccinated hamsters prior to start of the experiment).
8
SARS-CoV-2 Spike Protein IgG and IgM Detection
9
SARS-CoV-2 Spike Protein ELISA Protocol
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Stabilized SARS-CoV-2 spike protein was obtained from the Vaccine Research Centre, Bethesda, USA. Maxisorp plates (Nunc) were coated overnight at 4°C with 100 ng/well spike protein in PBS. Plates were blocked with 100 μl of casein in PBS (Thermo Fisher) for 1hr at RT. Serum serially diluted 2x in casein in PBS was incubated at RT for 1hr. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat-anti-monkey IgG (Seracare, 074–11-021) in casein and TMB 2-component peroxidase substrate (Seracare, 5120–0047), developed for 5–10 min, and reaction was stopped using stop solution (Seracare, 5150–0021) and read at 450 nm. All wells were washed 4x with PBST 0.1% tween in between steps. Threshold for positivity was set at 3x OD value of negative control (serum obtained from non-human primates prior to start of the experiment) or 0.2, whichever one was higher.
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