N. brasiliensis antigen-specific serum antibody isotypes and total IgE titers from infected mice were determined as follows. Blood was collected in serum separator tubes (BD Bioscience, San Diego, CA) and centrifuged at 8 000×g for 10 min at 4°C to separate serum. The flat-bottom 96-well plates were coated with 10 μg/ml NbAg, blocked with 2% (w/v) milk powder for 2 h at 37°C and samples were loaded and incubated overnight at 4°C. Alkaline phosphatase labeled secondary antibody was added and incubated for 2 h at 37°C. The plates were developed by addition of 4-nitrophenyl substrate (Sigma). The absorbance was read at 405 nm using VersaMax microplate spectrophotometer (Molecular Devices, Germany).
4 nitrophenyl substrate
Manufactured by Merck Group
The 4-nitrophenyl substrate is a laboratory reagent used in various analytical and biochemical applications. It is a colorimetric substrate that undergoes enzymatic hydrolysis, resulting in the formation of a yellow-colored product. The core function of this substrate is to serve as a detection or quantification tool for specific enzymatic activities, without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Serum separator tube, by BD (3 mentions)
Versamax microplate spectrophotometer, by Molecular Devices (2 mentions)
4 nitrophenyl butyrate, by Merck Group (1 mentions)
4 nitrophenyl acetate, by Merck Group (1 mentions)
4 nitrophenyl trimethylacetate, by Merck Group (1 mentions)
Versamax, by Molecular Devices (1 mentions)
4 protocols using 4 nitrophenyl substrate
1
Serum Antibody Isotypes in Infected Mice
2
Kinetic Analysis of 4-Nitrophenyl Substrates
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4-nitrophenyl substrate-specific activity was determined in 50 µL reactions containing 25 mM Tris pH 7.5, 250 mM NaCl, 1 mM MnCl2, 10% glycerol, 1 µM protein, and 1 mM 4-nitrophenyl substrate. The tested substrates, 4-nitrophenyl acetate (Sigma, N8130), 4-nitrophenyl butyrate (Sigma N9876), and 4-nitrophenyl trimethylacetate (Sigma 135046), were resuspended in acetonitrile at 100 mM. Reactions without 4-nitrophenyl substrate were preincubated at 37°C for 10 min prior to assay initiation via substrate addition. Conversion of 4-nitrophenyl substrates to 4-nitrophenol was tracked photometrically at 37°C and A405nm. Experiments were performed in triplicate with technical duplicates.
3
Quantification of Antigen-Specific Antibodies
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N. brasiliensis antigen-specific serum antibody isotypes and total IgE titres from infected mice were determined as previously described [66] (link). Briefly, blood was collected in serum separator tubes (BD Bioscience, San Diego, CA) and centrifuged at 8 000×g for 10 min at 4°C to separate serum. The flat-bottom 96-well plates were coated with 10 µg/ml somatic N. brasiliensis antigen (NAg), blocked with 2% (w/v) milk powder for 2 h at 37°C and samples were loaded and incubated overnight at 4°C. Alkaline phosphatase labelled secondary antibody was added and incubated for 2 h at 37°C. The plates were developed by addition of 4-nitrophenyl substrate (Sigma). The absorbance was read at 405 nm using VersaMax microplate spectrophotometer (Molecular Devices, Germany).
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N. brasiliensis antigen-specific serum antibody isotypes and total IgE titres from infected mice were determined as previously described [66] (link). Briefly, blood was collected in serum separator tubes (BD Bioscience, San Diego, CA) and centrifuged at 8 000×g for 10 min at 4°C to separate serum. The flat-bottom 96-well plates were coated with 10 µg/ml somatic N. brasiliensis antigen (NAg), blocked with 2% (w/v) milk powder for 2 h at 37°C and samples were loaded and incubated overnight at 4°C. Alkaline phosphatase labelled secondary antibody was added and incubated for 2 h at 37°C. The plates were developed by addition of 4-nitrophenyl substrate (Sigma). The absorbance was read at 405 nm using VersaMax microplate spectrophotometer (Molecular Devices, Germany).
4
Serum Antibody Isotypes and IgE in S. mansoni Infection
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S. mansoni antigen-specific serum antibody isotypes and total IgE titers from infected mice were determined as follows. Blood was collected in serum separator tubes (BD Bioscience, San Diego, CA) and centrifuged at 8 000×g for 10 min at 4°C to separate serum. The flat-bottom 96-well plates were coated with 10 μg/ml SEA, blocked with 2% (w/v) milk powder for 2 h at 37°C and samples were loaded and incubated overnight at 4°C. Alkaline phosphatase labeled secondary antibody was added and incubated for 2 h at 37°C. The plates were developed by addition of 4-nitrophenyl substrate (Sigma). The absorbance was read at 405 nm using VersaMax microplate spectrophotometer (Molecular Devices, Germany).
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