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P nitrophenyl phosphate substrate

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P-nitrophenyl phosphate substrate is a colorimetric substrate used in biochemical assays. It is used to detect and quantify the presence of enzymes that can catalyze the hydrolysis of phosphate esters, such as alkaline phosphatase.

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Lab products found in correlation

Elx800 universal microplate reader, by Agilent Technologies (3 mentions) Kc junior software, by Agilent Technologies (2 mentions) H5 mouse monoclonal antibody, by Southern Biotech (2 mentions) Versamax microplate reader, by Molecular Devices (2 mentions) Goat anti mouse igg, by Southern Biotech (1 mentions)

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Disodium p-nitrophenyl phosphate substrate (2 citations)

5 protocols using p nitrophenyl phosphate substrate

1

Quantification of Gonococcal Antibodies

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Samples of vaginal wash and serum were collected from the mice at the indicated time points.17 , 26 (link) Gonococcus-specific IgA, IgG, IgM, and IgG subclass antibodies IgG1, IgG2a, IgG2b, and IgG3 in vaginal washes and sera were measured by ELISA on plates coated with whole gonococci, using undiluted vaginal wash and 10-fold diluted serum as starting dilutions. 17 , 26 (link) Total IgA, IgG, and IgM concentrations in secretions were assayed by ELISA on plates coated with anti-IgA, -IgG, or -IgM antibodies (Southern Biotech, Birmingham, AL). H5 mouse monoclonal antibody (specific for N. gonorrhoeae porin serovar PIB3) or affinity-purified mouse IgA, IgG, and IgM (Southern Biotech) were used to establish standard curves. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-mouse IgA, IgG, IgM, IgG1, IgG2a, IgG2b, or IgG3 antibody (Southern Biotech) and p-nitrophenylphosphate substrate (Southern Biotech). Plates were read in a VersaMax microplate reader with SoftMax software (Molecular Devices, Sunnyvale, CA) or an ELX800 Universal microplate reader with KC Junior software (Bio-Tek Instruments, Winooski, VT). Antibody data were expressed as relative (fold increase) to the antibody levels detected in control samples (from sham-immunized mice) assayed simultaneously.
Liu Y., Hammer L.A., Liu W., Hobbs M.M., Zielke R.A., Sikora A.E., Jerse A.E., Egilmez N.K, & Russell M.W. (2017). Experimental Vaccine Induces Th1-driven Immune Responses and Resistance to Neisseria gonorrhoeae Infection in a Murine Model. Mucosal immunology, 10(6), 1594-1608.
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2

Gonococcus-specific Antibody Assays

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Samples of serum, vaginal wash, and saliva were collected from the mice 2 weeks after the last immunization, or at termination of the experiment after clearance of the infection (~2 weeks after inoculation) (10 (link), 33 (link)). Gonococcus-specific IgG and IgA antibodies in serum and vaginal washes were assayed by ELISA on plates coated with whole gonococci of the strain used for vaginal challenge unless stated otherwise (10 (link), 33 (link)). Total IgA and IgG concentrations in secretions were assayed by ELISA on plates coated with anti-IgA or anti-IgG antibodies (Southern Biotech, Birmingham, AL). H5 mouse monoclonal antibody (specific for N. gonorrhoeae porin serovar PIB3 in FA1090) was used to establish standard curves when IgG antibodies were being assayed against FA1090. Otherwise, affinity-purified mouse IgG or IgA (Southern Biotech) was used to establish standard curves on plates coated with anti-mouse IgG or IgA, respectively. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-mouse IgG or IgA antibody (Southern Biotech) and p-nitrophenyl phosphate substrate (Southern Biotech). Plates were read in an ELX800 Universal microplate reader with Gen 5 software (Bio-Tek Instruments, Winooski, VT).
Liu Y., Hammer L.A., Daamen J., Stork M., Egilmez N.K, & Russell M.W. (2022). Microencapsulated IL-12 Drives Genital Tract Immune Responses to Intranasal Gonococcal Outer Membrane Vesicle Vaccine and Induces Resistance to Vaginal Infection with Diverse Strains of Neisseria gonorrhoeae. mSphere, 8(1), e00388-22.
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3

ELISA and Immunoblotting for C. rodentium-specific Ig

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For measurement of C. rodentium-specific Ig by ELISA, 96-well ELISA plates were coated with heat-killed C. rodentium. Diluted serum or luminal content were then added to the coated plate, and the presence of C. rodentium-specific Igs was detected by alkaline phosphatase-conjugated polyclonal goat anti-mouse IgG, IgM or IgA Abs (Southern Biotechnology Associates, Birmingham, AL). Plates were developed using p-nitrophenyl phosphate substrate (Southern Biotechnology Associates) and OD405 values determined. For detection of C. rodentium-specific Ig by immunoblotting, heat-killed WT C. rodentium, Δler mutant strain or purified C. rodentium Intimin were loaded with 12% SDS-PAGE, and proteins detected with anti-mouse IgG antibody and enhanced chemiluminescent substrate (Thermo Scientific).
Kamada N., Sakamoto K., Seo S.U., Zeng M.Y., Kim Y.G., Cascalho M., Vallance B.A., Puente J.L, & Núñez G. (2015). Humoral Immunity in the Gut Selectively Targets Phenotypically Virulent Attaching-and-Effacing Bacteria for Intraluminal Elimination. Cell host & microbe, 17(5), 617-627.
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4

Quantification of Gonococcal Antibodies

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Samples of vaginal wash and serum were collected from the mice at the indicated time points.17 , 26 (link) Gonococcus-specific IgA, IgG, IgM, and IgG subclass antibodies IgG1, IgG2a, IgG2b, and IgG3 in vaginal washes and sera were measured by ELISA on plates coated with whole gonococci, using undiluted vaginal wash and 10-fold diluted serum as starting dilutions. 17 , 26 (link) Total IgA, IgG, and IgM concentrations in secretions were assayed by ELISA on plates coated with anti-IgA, -IgG, or -IgM antibodies (Southern Biotech, Birmingham, AL). H5 mouse monoclonal antibody (specific for N. gonorrhoeae porin serovar PIB3) or affinity-purified mouse IgA, IgG, and IgM (Southern Biotech) were used to establish standard curves. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-mouse IgA, IgG, IgM, IgG1, IgG2a, IgG2b, or IgG3 antibody (Southern Biotech) and p-nitrophenylphosphate substrate (Southern Biotech). Plates were read in a VersaMax microplate reader with SoftMax software (Molecular Devices, Sunnyvale, CA) or an ELX800 Universal microplate reader with KC Junior software (Bio-Tek Instruments, Winooski, VT). Antibody data were expressed as relative (fold increase) to the antibody levels detected in control samples (from sham-immunized mice) assayed simultaneously.
Liu Y., Hammer L.A., Liu W., Hobbs M.M., Zielke R.A., Sikora A.E., Jerse A.E., Egilmez N.K, & Russell M.W. (2017). Experimental Vaccine Induces Th1-driven Immune Responses and Resistance to Neisseria gonorrhoeae Infection in a Murine Model. Mucosal immunology, 10(6), 1594-1608.
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5

ELISA Antibody Titer Quantification

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Serum samples were obtained from blood collected from the submandibular vein at the indicated timepoints. Plates were coated with either goat anti-mouse IgG (SouthernBiotech), NP30-BSA and NP7-BSA (Biosearch Technologies), or heat-inactivated IAVs overnight at 4°C. Serum samples underwent twofold serial dilutions starting from the indicated initial dilution. Bound antibodies were detected using alkaline phosphatase–conjugated anti-IgG1/2b/2c/3, IgM or IgA, and p-nitrophenyl phosphate substrate (SouthernBiotech). The reaction was stopped by adding 1.5 N NaOH solution, and optical density was measured at 405 nm.
Panneton V., Mindt B.C., Bouklouch Y., Bouchard A., Mohammaei S., Chang J., Diamantopoulos N., Witalis M., Li J., Stancescu A., Bradley J.E., Randall T.D., Fritz J.H, & Suh W.K. (2023). ICOS costimulation is indispensable for the differentiation of T follicular regulatory cells. Life Science Alliance, 6(4), e202201615.
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