Nunc MaxiSorp plates were coated with 50 μl/well of 4 μg/ml anti-MPO antibody (R&D Systems) in PBS overnight at 4°C. Plates were washed three to five times with PBS/0.05% Tween and blocked with PBS/1% BSA for 1.5 h at room temperature. Plates were washed and 50 μl of undiluted plasma samples were added to each well and incubated overnight at 4°C. Plates were washed three to five times with PBS/0.05% Tween, and 50 μl of mouse anti-dsDNA (1:2,000; Abcam) diluted in 1× PBS/1% BSA was added and incubated overnight at 4°C. Plates were washed and alkaline phosphatase–conjugated anti-mouse IgG Fcγ2a (1:5,000; Jackson Immunoresearch) diluted in PBS/1% BSA was added and incubated for 3 h at 4°C, followed by development with diethanolamine substrate buffer (Thermo Fisher Scientific) and p-nitrophenyl phosphate substrate tablets (Sigma-Aldrich). Undiluted bronchoalveolar lavage fluid from mice with lung neutrophilia (Fogli et al., 2013 (link)) was used as a positive control on the same plate.
P nitrophenyl phosphate substrate tablets
Manufactured by Merck Group
P-nitrophenyl phosphate substrate tablets are a laboratory reagent used in various biochemical and analytical applications. These tablets contain the compound p-nitrophenyl phosphate, which is a colorless substrate that can be enzymatically hydrolyzed to produce a yellow-colored product. This reaction can be used to measure the activity of certain enzymes, such as alkaline phosphatase, in a variety of sample types.
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2 protocols using p nitrophenyl phosphate substrate tablets
1
Enzyme-linked Immunoassay for Anti-MPO Antibodies
2
ELISA for HA and Virus Binding
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96-well plates were coated with 500 ng per well of recombinant purified HA proteins, at 4 °C during 16 h. Alternatively, plates were coated with concentrated viruses. To this end, clarified supernatants from infected cells were concentrated 100X by treating them with a polyethylene glycol virus precipitation solution (System Biosciences). Then, protein concentration was quantified and each well was coated with 50 μl of a 10 μg/mL solution. After washing with PBS containing 0.1% Tween 20, the coated wells were blocked with PBS containing 2.5% BSA, and then the plates were incubated with 1:2 dilutions of human serum (starting dilution, 1:10) for 2 h at 37 °C, or with mAbs (15 μg/mL) overnight at 4 °C. Then, the wells were washed with PBS containing 0.1% Tween 20, and incubated with alkaline phosphatase-conjugated goat anti-human IgG (Southern Biotechnology) for 30 min at 37 °C. The reactions were developed with p-nitrophenyl phosphate substrate tablets (Sigma) in diethanolamine buffer for 30 min at room temperature, and read at 405 nm (Vmax kinetic microplate reader; Molecular Devices).
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