The enzyme-linked immunosorbent assay (ELISA) used to detect nucleocapsid protein (N) specific antibodies (N-ELISA) was described previously [34 (link)]. Species-specific secondary antibodies were used at the following concentrations: peroxide-labeled anti-hamster (1:10,000) (Sera Care, Gaithersburg, MD), peroxide-labeled anti-ferret (1:5,000) (Sigma Aldrich, St. Louis, MO), and alkaline phosphatase conjugated anti-monkey (1:1,000) (MilliporeSigma, St. Louis, MO). Assays using peroxide labeled antibodies were developed with TMB microwell peroxidase substrate (Sera Care) at an absorbance of 450 nm; assays using alkaline phosphatase conjugated antibodies were developed with p-nitrophenyl phosphate (PNPP) (ThermoFisher Scientific, Waltham, MA) at 405 nm. A sample was considered positive if its peak optical density (OD) value was greater than either 0.025 or the background value (the average of three negative controls + 3 times their standard deviation), whichever was higher. The specific OD sum is the summation of all values greater than background and represents the area under the titer curve.
P nitrophenyl phosphate pnpp
Manufactured by Thermo Fisher Scientific
Sourced in United States
P-nitrophenyl phosphate (PNPP) is a colorimetric substrate used in various biochemical and analytical applications. It is used to detect and measure the activity of enzymes that catalyze the hydrolysis of phosphate esters, such as alkaline phosphatase. When PNPP is hydrolyzed by the enzyme, it produces a yellow-colored product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Transfection reagent, by Promega (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Alkaline phosphatase conjugated anti monkey, by Merck Group (1 mentions)
Peroxide labeled anti hamster, by LGC (1 mentions)
Tmb microwell peroxidase substrate, by LGC (1 mentions)
El311sx, by Agilent Technologies (1 mentions)
Biotinylated goat anti mouse igg, by Southern Biotech (1 mentions)
7 protocols using p nitrophenyl phosphate pnpp
1
ELISA for Detecting Antibodies to N Protein
2
ELISA for Catfish Antibodies
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To assay antibodies in catfish serum and skin mucus, ELISA was performed using E. piscicida LPS following the protocol previously described [29 (link)]. In brief, E. piscicida LPS (100 ng/well) in coating buffer was used to coat the polystyrene 96-well flat-bottom microtiter plates (Dynatech Laboratories Inc., Chantilly, VA, USA). Wells were blocked by adding 300 μL of 5% bovine serum albumin (BSA). Diluted samples of catfish serum or 100 μL of mucus was added to wells in duplicate, while in blank control wells, 100 µL of sterile PBS was added. Moreover, 100 μL of diluted mouse anti-catfish IgM monoclonal antibody (Aquatic Diagnostics Ltd., Scotland, UK) was added to each well. Biotinylated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL, USA) was diluted, and 100 μL of it was added to the wells. For the color development, p-nitrophenyl phosphate (PNPP, Thermo Fisher Scientific, Rockford, IL, USA) was added, and the OD was noted at 405 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT, USA).
3
Quantifying ADAM17 Shedding Activity
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Shedding activity of ADAM17 variants was measured by an alkaline phosphatase (AP)‐based assay. 24‐well plates were first coated with Poly‐l ‐lysine (Sigma). Around 4 × 105 cells/well were plated onto 24‐well plates containing a mix of ADAM17 variants with eiger fused to AP plasmids and transfection reagent (Fugene). Media was changed after 24 h. 48 h, post‐plating cells were treated with only 100 nM PMA, 100 nM PMA combined with 10 μM TAPI‐1 or treated with the corresponding volume of vehicle (DMSO). Cells were incubated for 120 min at 37°C. Cell supernatants were collected, and the cells were washed in PBS and lysed in 200 μl Triton X‐100 lysis buffer. The activity of AP in cell lysates and supernatants was determined by incubating 100 μl AP substrate p‐nitrophenyl phosphate (PNPP) (Thermo Scientific) with 100 μl cell lysate or cell supernatant at room temperature followed by the measurement of the absorption at 405 nm. The percentage of AP‐conjugated material released from each well was calculated by dividing the signal from the supernatant by the sum of the signal from lysate and supernatant. The data were expressed as mean of at least three independent experiments.
4
ADAM17 Shedding Activity Assay
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Shedding activity of ADAM17 variants was measured by an alkaline phosphatase (AP)‐based assay. For this assay, 5 × 106 cells were seeded on a 10‐cm dish and transiently transfected with the ADAM17 substrate IL‐1RII fused to an alkaline phosphatase (AP). Transient transfection was performed via the use of Lipofectamine 3000 (Thermo Fischer Scientific). After 24 h, 2 × 105 cells/well were transferred into 24‐well plates. 24 h later, cells were treated with only 100 nM PMA, 100 nM PMA combined with 10 μM TAPI‐1 or treated with the corresponding volume of vehicle (DMSO). Cells were incubated for 120 min at 37°C. The activity of AP in cell lysates and supernatants was determined by incubating 100 μl AP substrate p‐nitrophenyl phosphate (PNPP) (Thermo Scientific) with 100 μl cell lysate or cell supernatant at room temperature followed by the measurement of the absorption at 405 nm. The percentage of AP‐conjugated material released from each well was calculated by dividing the signal from the supernatant by the sum of the signal from lysate and supernatant. The data were expressed as mean of at least three independent experiments. All data points, including outliers, were used for statistical analysis.
5
Enzymatic Assay with p-NPP
6
ADAM17 Shedding Activity Assay
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Shedding activity of ADAM17 variants was measured by an alkaline phosphatase (AP)-based assay. For this assay 5x10 6 cells were seeded on a 10-cm dish, and transiently transfected with the ADAM17 substrate IL-1RII fused to an alkaline phosphatase (AP). Transient transfection was performed via the use of Lipofectamine 3000 (Thermo Fischer Scientific). After 24 hours, 2x10 5 cells/well were transferred into 24-well plates. 24 hour later, cells were treated with only 100 nM PMA, 100 nM PMA combined with 10 µM TAPI-1 or treated with the corresponding volume of vehicle (DMSO). Cells were incubated for 120 minutes at 37 °C. The activity of AP in cell lysates and supernatants was determined by incubating 100 µl AP substrate p-nitrophenyl phosphate (PNPP) (Thermo Scientific) with 100 µl cell lysate or cell supernatant at room temperature followed by the measurement of the absorption at 405 nm. The percentage of AP-conjugated material released from each well was calculated by dividing the signal from the supernatant by the sum of the signal from lysate and supernatant. The data was expressed as mean of at least three independent experiments.
7
Quantifying ADAM17 Shedding Activity
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Shedding activity of ADAM17 variants was measured by an alkaline phosphatase (AP)-based assay. 24 well plates were first coated with Poly-L-lysine (Sigma).
Around 4x10 5 cells/well were plated onto 24-well plates containing a mix of ADAM17 variants with eiger fused to AP plasmids and transfection reagent (Fugene). Media was changed after 24 hours. 48 hours, post plating cells were treated with only 100 nM PMA, 100 nM PMA combined with 10 µM TAPI-1 or treated with the corresponding volume of vehicle (DMSO). Cells were incubated for 120 minutes at 37 °C. Cell supernatants were collected, the cells were washed in PBS and lysed in 200 µl Trition X-100 lysis buffer. The activity of AP in cell lysates and supernatants was determined by incubating 100 µl AP substrate p-nitrophenyl phosphate (PNPP) (Thermo Scientific) with 100 µl cell lysate or cell supernatant at room temperature followed by the measurement of the absorption at 405 nm. The percentage of APconjugated material released from each well was calculated by dividing the signal from the supernatant by the sum of the signal from lysate and supernatant. The data was expressed as mean of at least three independent experiments.
Around 4x10 5 cells/well were plated onto 24-well plates containing a mix of ADAM17 variants with eiger fused to AP plasmids and transfection reagent (Fugene). Media was changed after 24 hours. 48 hours, post plating cells were treated with only 100 nM PMA, 100 nM PMA combined with 10 µM TAPI-1 or treated with the corresponding volume of vehicle (DMSO). Cells were incubated for 120 minutes at 37 °C. Cell supernatants were collected, the cells were washed in PBS and lysed in 200 µl Trition X-100 lysis buffer. The activity of AP in cell lysates and supernatants was determined by incubating 100 µl AP substrate p-nitrophenyl phosphate (PNPP) (Thermo Scientific) with 100 µl cell lysate or cell supernatant at room temperature followed by the measurement of the absorption at 405 nm. The percentage of APconjugated material released from each well was calculated by dividing the signal from the supernatant by the sum of the signal from lysate and supernatant. The data was expressed as mean of at least three independent experiments.
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