Mice were injected intraperitoneally with 50 μg TNP-LPS or 150 μg adsorbed NP-KLH (Biosearch Technology) 1:1 ratio onto Alu-Gel-S (Serva). Spleens and BM were taken after the indicated time points p.i., and PCs were analyzed by flow cytometry. Blood samples were taken at the indicated time points p.i. TNP-specific antibodies were detected by ELISA, using TNP-BSA (10 µg/ml) for capture and biotinylated anti-mouse IgM (Southern Biotech), IgG1 (Southern Biotech), and IgG3 (BD Bioscience) for detection. Mouse sera were serially diluted in duplicate with an appropriate standard (mouse a-TNP-IgM; 55581, BD Pharmingen, or a reference sample). ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma-Aldrich) and phosphorylated nitrophenyl phosphate (Sigma-Aldrich). Absorbance at 405 nm was determined with a SPECTRAmax 250 plate reader (Molecular Device).
Phosphorylated nitrophenyl phosphate
Manufactured by Merck Group
Phosphorylated nitrophenyl phosphate is a colorimetric substrate used in biochemical assays. It is a phosphate ester that undergoes hydrolysis in the presence of phosphatase enzymes, resulting in the release of nitrophenol, which can be detected spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin alkaline phosphatase, by Merck Group (5 mentions)
Biotinylated anti mouse igm and igg, by Southern Biotech (2 mentions)
Np23 bsa, by Southern Biotech (2 mentions)
Np3 bsa, by Southern Biotech (2 mentions)
Spectramax 190 plate reader, by Molecular Devices (2 mentions)
Alu gel s, by Serva Electrophoresis (1 mentions)
Spectramax 250 plate reader, by Molecular Devices (1 mentions)
Spectramax 190 plate, by Molecular Devices (1 mentions)
Igg2c, by Southern Biotech (1 mentions)
Igg2b, by Southern Biotech (1 mentions)
Igg2c, by Fortis Life Sciences (1 mentions)
Np lps, by Biosearch Technologies (1 mentions)
Np 23 klh, by Biosearch Technologies (1 mentions)
Elisa plate, by Thermo Fisher Scientific (1 mentions)
6 protocols using phosphorylated nitrophenyl phosphate
1
Quantifying TNP-specific Antibody Responses
2
Vaccinia Virus Infection and NP-Specific Antibody Response in Mice
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For immunization, mice were injected intra-peritoneally with 50 μg NP23-KLH (Biosearch Technology) in 4 mg Alum (ThermoScientific). Blood samples were taken from the lateral tail-vein on day 0, 3, 7, 13, 28 after immunization. For infection, 104 PFU of Vaccinia Virus Western Reserve strain (vacv) was injected into isoflurane-anesthetized animals in the footpads. NP-specific antibody titers were detected by ELISA, using NP23-BSA, NP3-BSA, and biotinylated anti–mouse IgM and IgG (Southern Biotech). Titers were determined from the dilution curve in the linear range of absorbance. All non-commercial ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma) and phosphorylated nitrophenyl-phosphate (Sigma). Absorbance at 405 nm was determined with a SPECTRAmax190 plate reader (Molecular Devices). NP-specific antibody-secreting cells and vacv-specific antibody-secreting cells were captured using activated ELISPOT plates coated with either NP23-BSA or vacv respectively. Detection was made using biotinylated anti–mouse IgM (Southern Biotech), IgG (Southern Biotech). All ELISPOT plates were developed with alkaline-phosphatase streptavidin (Sigma) and the BCiP®/NBT reaction (Sigma). Images were acquired using CTL 4.0 (ImmunoSpot®).
3
Characterization of NP-specific Antibody Response
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Mice were injected intraperitoneally (i.p.) with 50 μg of NP33-KLH (Biosearch Technology) in 4 mg of alum (Thermo Scientific). Blood samples were taken from the lateral tail vein on days 0, 4, 7, 14, and 21 after immunization. NP-specific antibody titers were detected by ELISA, using NP29-BSA or NP4-BSA for capture and biotinylated anti-mouse IgM (Southern Biotech), IgG (Southern Biotech), IgG1 (Southern Biotech), and IgG3 (BD Biosciences) for detection. Titers were determined from the dilution curve in the linear range of absorbance. ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma) and phosphorylated nitrophenyl phosphate (Sigma). Absorbance at 405 nm was determined with a SPECTRAmax190 plate reader (Molecular Devices).
4
Immunization and Infection Protocol
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For immunization, mice were injected i.p. with 50 µg NP19-KLH (Biosearch Technology) in 4mg Alum (Thermo Fisher Scientific). Blood samples were taken from the lateral tail vein on day 0, 7, 14, 21, 28 after immunization.
For infection, mice were intranasally immunized with 200pfu of Influenza A virus (PR8 strain). Blood samples were taken from the lateral tail vein on day 0 and 9 after infection.
NP- or Influenza-specific antibody titers were detected by ELISA, using NP23-BSA, NP3-BSA, or live Influenza virus for capture, and biotinylated anti–mouse IgM (Southern Biotech), IgG (Southern Biotech), IgG2b (Southern Biotech), IgG2c (Southern Biotech), and IgG3 (BD). Titers were determined from the dilution curve in the linear range of absorbance.
Total antibody levels in serum before immunization were measured by sandwich ELISA. For capture, anti-IgM II/41 (BD), anti-IgG2b (BD), and anti-IgG2c (BD) were used. Purified IgM (BD), IgG2b (Southern Biotech), or IgG2c (Bethyl) were used as standards. Biotinylated antibodies were used for detection. For IgG1, a commercial kit was used (eBioscience).
All noncommercial ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma-Aldrich) and phosphorylated nitrophenyl phosphate (Sigma-Aldrich). Absorbance at 405 nm was determined with a SPECTRAmax190 plate reader (Molecular Devices).
For infection, mice were intranasally immunized with 200pfu of Influenza A virus (PR8 strain). Blood samples were taken from the lateral tail vein on day 0 and 9 after infection.
NP- or Influenza-specific antibody titers were detected by ELISA, using NP23-BSA, NP3-BSA, or live Influenza virus for capture, and biotinylated anti–mouse IgM (Southern Biotech), IgG (Southern Biotech), IgG2b (Southern Biotech), IgG2c (Southern Biotech), and IgG3 (BD). Titers were determined from the dilution curve in the linear range of absorbance.
Total antibody levels in serum before immunization were measured by sandwich ELISA. For capture, anti-IgM II/41 (BD), anti-IgG2b (BD), and anti-IgG2c (BD) were used. Purified IgM (BD), IgG2b (Southern Biotech), or IgG2c (Bethyl) were used as standards. Biotinylated antibodies were used for detection. For IgG1, a commercial kit was used (eBioscience).
All noncommercial ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma-Aldrich) and phosphorylated nitrophenyl phosphate (Sigma-Aldrich). Absorbance at 405 nm was determined with a SPECTRAmax190 plate reader (Molecular Devices).
5
NP-specific Antibody Response Assay
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For T-independent immunisation, mice were injected intraperitoneally with 2 μg of NP-LPS (Biosearch Technologies). Blood samples were taken from the lateral tail vein on days –1, 3, 6, and 9. For T-dependent immunisation, the mice were injected intraperitoneally with 50 μg NP23-KLH (Biosearch Technologies) in 4 mg alum (Thermo Fisher Scientific). Blood samples were taken from the lateral tail vein on days –1, 3, 7, 14, 21, 28, and 69. In both cases, NP-specific antibody titres were detected by ELISA, using NP23-BSA, NP3-BSA (for T-dependent immunisation), and biotinylated anti-mouse IgM and IgG (Southern Biotech). Titres were determined from the dilution curve in the linear range of absorbance. All noncommercial ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma-Aldrich) and phosphorylated nitrophenyl-phosphate (Sigma-Aldrich). Absorbance at 405 nm was determined with a SPECTRAmas190 plate reader (Molecular Devices).
6
Evaluating Anti-MUC1 Antibody Responses
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All experiments were routinely performed in groups of eight mice. On days 0, 14 and 28, the mice were intraperitoneally (i.p.) administered PBS, 20 µg MUC1 peptide, 8.5 µg T7 or 28 µg T7-MUC1. A total of 0.2 ml blood was collected from the tail of the mice 7 days after the last vaccination, and serum was harvested from the blood for serologic assays. Anti-MUC1 IgG, IgG1, IgG2a and IgM antibody titers were determined by ELISA as previously described (18 (link)). ELISA plates (Thermo Fisher Scientific, Inc.) were coated with the MUC1 peptide, and serial dilutions of the serum were allowed to bind to immobilized MUC1. Detection was performed by adding alkaline phosphatase-conjugated anti-mouse antibodies and phosphorylated nitrophenyl phosphate (Sigma-Aldrich; Merck KGaA), and the optical density was measured at 405 nm using a microplate reader.
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