Plasma samples collected before vaccination, 21-28 days after the first vaccination, and then 14-30 days after the second vaccination from each subject were titrated in 96-well plates coated with 3 μg/ml S1-RBD in PBS and blocked with 1% BSA. Plate-bound Ig of all isotypes was detected by incubating the wells sequentially with horseradish peroxidase-labeled antibodies specific for human Ig heavy and light chains (Invitrogen/ThermoFisher #31412), and KPL ABTS Peroxidase Substrate (SeraCare #5120-0043). Each plate contained the same known positive and negative serum as a reference. The optical density (405 nm) of each well was measured in an ELISA plate reader. Titers were calculated as the reciprocal of the dilution that gave a half-maximal optical density (405) nm value.
Kpl abts peroxidase substrate
Manufactured by LGC
The KPL ABTS® peroxidase substrate is a colorimetric reagent used to detect and quantify the presence of peroxidase enzymes in biological samples. It provides a sensitive and stable chromogenic reaction that can be measured spectrophotometrically.
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Lab products found in correlation
5 protocols using kpl abts peroxidase substrate
1
SARS-CoV-2 Antibody Response Quantification
2
IFN-γ Neutralization Assay
3
IFN-γ Neutralization Assay
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To neutralize IFN-γ, 1mg anti-IFN-γ (XMG1.2, BioXcell Cat #: BE0055) was given intraperitoneally (i.p.) 1 day prior and 2 days post infection with maCp. To also neutralize IL-12 or IL-18, 2mg anti-IL-12p40 (C17.8 BioXcell Cat #: BE0051) or 1mg anti-IL-18 (YIGIF74-1G7, BioXcell Cat #: BE0237) were given i.p. on days −4, −1, and 1, while anti-IFN-γ was given on day −2 and day 2. Intestinal IFN-γ levels were assessed from 5mm biopsy punches that were incubated in complete RPMI at 37°C for 24 hours. Clear, flat-bottom 96-well plates (Immunulon 4 HBX) were coated with 0.25μg/mL anti-IFN-γ (AN-18, Invitrogen Ref #:14-7313-85) at 4°C overnight. Samples were added and IFN-γ left to bind at 37°C for 2 hours. 0.25μg/mL biotinylated anti-IFN-γ (R4-6A2, eBioscience Ref #: 13-7312-85) in PBS with 2.5% FBS and 0.05% Tween was added for 1 hour at room temperature, followed by peroxidase-labeled streptavidin for 30 minutes. Finally, KPL ABTS® peroxidase substrate (SeraCare Cat #: 5120-0041) was applied for detection.
4
ELISA for SARS-CoV-2 Antibody Titers
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Plasma samples were serially diluted in 96-well plates coated with 3 μg/mL S1-RBD in PBS and blocked with 1% BSA. Plates were incubated at 37°C for 1 hour with horseradish peroxidase–labeled antibodies specific for human Ig heavy and light chains (Invitrogen/Thermo Fisher Scientific, 31412) and developed with KPL ABTS Peroxidase Substrate (SeraCare, 5120-0043). Each plate contained a positive and negative serum as a reference. An ELISA plate reader was used to measure the optical density (405 nm) of each well. Titers were calculated as the reciprocal of the dilution that gave a half-maximal optical density value.
5
Neutralizing IFN-γ, IL-12, and IL-18 in Cryptosporidium infection
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To neutralize IFN-g, 1mg anti-IFN-g (XMG1.2, BioXcell Cat #: BE0055) was given intraperitoneally (i.p.) 1 day prior and 2 days post infection with C. tyzzeri. To also neutralize IL-12 or IL-18, 2mg anti-IL-12p40 (C17.8 BioXcell Cat #: BE0051) or 1mg anti-IL-18 (YIGIF74-1G7, BioXcell Cat #: BE0237) were given i.p. on days -4, -1, and 1, while anti-IFN-g was given on day -2 and day 2. Intestinal IFN-g levels were assessed from 5mm biopsy punches that were incubated in complete RPMI at 37°C for 24 hours. Clear, flat-bottom 96-well plates (Immunulon 4 HBX) were coated with 0.25µg/mL anti-IFN-g (AN-18, Invitrogen Ref #:14-7313-85) at 4°C overnight. Samples were added and IFN-g left to bind at 37ºC for 2 hours. 0.25µg/mL biotinylated anti-IFN-g (R4-6A2, eBioscience Ref #: 13-7312-85) in PBS with 2.5% FBS and 0.05% Tween was added for 1 hour at room temperature, followed by peroxidase-labeled streptavidin for 30 minutes. Finally, KPL ABTS® peroxidase substrate (SeraCare Cat #: 5120-0041) was applied for detection.
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