Serum samples from SARS-CoV-2-infected animals were inactivated by γ-irradiation and used in BSL2 according to IBC-approved SOPs. NUNC Maxisorp Immuno plates were coated with 50 μl of 1 μg/mL of recombinant SARS-CoV-2 S (S1+S2) antigen (Sino Biological) at 4 °C overnight and then washed three times with PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 3% skim milk in PBS for 1 h at room temperature, followed by three additional washes with PBST. The plates were incubated with 50 μl of serial dilutions of the samples in PBS containing 1% skim milk for 1 h at room temperature. After three washes with PBST, the bound antibodies were labeled using 50 μl of 1:2500 peroxidase anti-hamster IgG (H+L) (SeraCare Life Sciences, cat#5220-0371) diluted in 1% skim milk in PBST. After incubation for 1 h at room temperature and three washes with PBST, 50 μl of KPL ABTS peroxidase substrate solution mix (SeraCare Life Sciences) was added to each well, and the mixture was incubated for 30 min at room temperature. The optical density (OD) at 405 nm was measured using a GloMax® explorer (Promega). The OD values were normalized to the baseline samples obtained with naïve hamster serum and the cutoff value was set as the mean OD plus standard deviation of the blank.
Kpl abts peroxidase substrate solution mix
Manufactured by LGC
KPL ABTS peroxidase substrate solution mix is a liquid substrate for the detection of peroxidase-labeled conjugates in enzyme-linked immunoassays (ELIAs) and other peroxidase-based applications. The solution contains the necessary components for the colorimetric detection of peroxidase activity, providing a stable and consistent reaction.
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5 protocols using kpl abts peroxidase substrate solution mix
1
SARS-CoV-2 Antibody Detection Assay
2
Quantifying SARS-CoV-2 Antibodies in Hamsters
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Serum samples from SARS-CoV-2 infected animals were inactivated by γ-irradiation and used in BSL2 according to IBC-approved SOPs. NUNC Maxisorp Immuno plates were coated with 50 μl of 1 μg/mL of recombinant SARS-CoV-2 S (S1+S2), SARS-CoV-2 RBD (Sino Biological) or EBOV GP at 4°C overnight and then washed three times with PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 3% skim milk in PBS for 3 hours at room temperature, followed by three additional washes with PBST. The plates were incubated with 50 μl of serial dilutions of the samples in PBS containing 1% skim milk for 1 hour at room temperature. After three washes with PBST, the bound antibodies were labeled using 50 μl of 1:2,500 peroxidase anti-hamster IgG (H+L) (SeraCare Life Sciences) diluted in 1% skim milk in PBST. After incubation for 1 h at room temperature and three washes with PBST, 50 μl of KPL ABTS peroxidase substrate solution mix (SeraCare Life Sciences) was added to each well, and the mixture was incubated for 30 min at room temperature. The optical density (OD) at 405 nm was measured using a GloMax® explorer (Promega) plate reader. The OD values were normalized to the baseline samples obtained with naïve hamster serum and the cutoff value was set as the mean OD plus standard deviation of the blank.
3
SARS-CoV-2 Antibody ELISA Assay
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Serum samples from SARS-CoV-2 infected animals were inactivated by γ-irradiation and used in BSL2 according to IBC-approved SOPs. NUNC Maxisorp Immuno plates were coated with 50 μl of 1 μg/mL of recombinant SARS-CoV-2 S (S1+S2), SARS-CoV-2 RBD (Sino Biological) or EBOV GP (32 (link)) at 4°C overnight and then washed three times with PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 3% skim milk in PBS for 3 hours at room temperature, followed by three additional washes with PBST. The plates were incubated with 50 μl of serial dilutions of the samples in PBS containing 1% skim milk for 1 hour at room temperature. After three washes with PBST, the bound antibodies were labeled using 50 μl of 1:2,500 peroxidase anti-hamster IgG (H+L) (SeraCare Life Sciences) diluted in 1% skim milk in PBST. After incubation for 1 h at room temperature and three washes with PBST, 50 μl of KPL ABTS peroxidase substrate solution mix (SeraCare Life Sciences) was added to each well, and the mixture was incubated for 30 min at room temperature. The optical density (OD) at 405 nm was measured using a GloMax® explorer (Promega) plate reader. The OD values were normalized to the baseline samples obtained with naïve hamster serum and the cutoff value was set as the mean OD plus standard deviation of the blank.
4
SARS-CoV-2 Antibody Detection Assay
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Serum samples from SARS-CoV-2-infected animals were inactivated by gamma-irradiation and used in BSL2 according to IBC-approved SOPs. NUNC Maxisorp Immuno plates were coated with 50 μl of 1 μg/mL of recombinant SARS-CoV-2 S (S1+S2) antigen at 4°C overnight and then washed three times with PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 3% skim milk in PBS for 1 hour at room temperature, followed by three additional washes with PBST. The plates were incubated with 50 μl of serial dilutions of the samples in PBS containing 1% skim milk for 1 hour at room temperature. After three washes with PBST, the bound antibodies were labeled using 50 μl of 1:2,500 peroxidase anti-hamster IgG (H+L) (SeraCare Life Sciences) diluted in 1% skim milk in PBST. After incubation for 1 hour at room temperature and three washes with PBST, 50 μl of KPL ABTS peroxidase substrate solution mix (SeraCare Life Sciences) was added to each well, and the mixture was incubated for 30 min at room temperature. The optical density (OD) at 405 nm was measured using a GloMax® explorer (Promega). The OD values were normalized to the baseline samples obtained with naïve hamster serum and the cutoff value was set as the mean OD plus standard deviation of the blank.
5
EBOV sGP Sandwich ELISA Protocol
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NUNC Maxisorp Immunoplates were coated with 50 μL of 1 μg/mL ZGP42/3.7 at 4 °C overnight and then washed once with PBS containing 0.05% Tween 20 (PBST). The plate was blocked with 3% skim milk (190 μL/well) for 3 h at room temperature. After washing once with PBST, 50 μL of appropriately diluted serum or plasma samples or the concentrated sGPs in PBS containing 1% skim milk were added and incubated overnight at 4 °C. After washing 3 times with PBST, 50 μL of 1 μg/mL Rabbit anti-EBOV sGP pAb (IBT BIOSERVICES; 0365-001) was added and incubated for 1 h at room temperature. After washing 3 times with PBST, the bound antibodies were labeled by using 50 μL of 1:1000 peroxidase AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch) diluted in 1% skim milk in PBST. After incubation for 1 h at room temperature and 3 PBST washes, 50 μL of KPL ABTS Peroxidase substrate solution mix (SeraCare Life Sciences) was added to each well, and the mixture was incubated for 15 min at room temperature. The optical density (OD) at 405 nm was measured. The standard curve for the sGP sandwich ELISA was carried out using serial dilutions of recombinant EBOV-sGP.
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