Kpl tmb 2 component peroxidase substrate kit

Manufactured by LGC

Sourced in United States

The KPL TMB 2-component peroxidase substrate kit is a laboratory reagent used as a chromogenic substrate for the detection and quantification of peroxidase enzymes in various applications, such as immunoassays and enzyme-linked immunosorbent assays (ELISAs). The kit contains two separate components that are mixed together prior to use to produce a colored reaction product.

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Lab products found in correlation

Kpl stop solution, by LGC (14 mentions) Maxisorp plate, by Thermo Fisher Scientific (14 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (12 mentions) Goat anti hamster igg fc, by Abcam (5 mentions) Blocker casein, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

TMB 2-component peroxidase substrate TMB substrate (KPL, KPL peroxidase substrate TMB peroxidase substrate TMB substrate

14 protocols using kpl tmb 2 component peroxidase substrate kit

ELISA Assay for SARS-CoV-2 Antibodies

Cited 1 time

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ELISAs were
performed as previously described.^{22 (link)} In
brief, Maxisorp plates (Nunc) were coated with 50 ng of spike protein
(Sinobiological SARS-CoV-2 (2019-nCoV) spike S1 + S2 ECD-HIS recombinant
protein (catalog #40589-V08B1)) per well. Plates were incubated overnight
at 4 °C. Plates were blocked with Blocker casein in phosphate
buffered saline (PBS) (ThermoFisher) for 1 h at room temperature (RT).
Hamster serum collected on day 14 post exposure was diluted 1:400
in Blocker casein in PBS and incubated for 1 h at RT. Serum from a
subset of positive qRT-PCR animals was titrated via a 2× serial
dilution to obtain antibody titers of positive animals. Secondary
goat antihamster IgG Fc (horseradish peroxidase-conjugated, Abcam)
spike-specific antibodies were used for detection and visualized with
a KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120-0047).
The reaction was stopped with KPL stop solution (SeraCare), and the
plates were read at 450 nm. The threshold for positivity was calculated
as the average plus 3 × the standard deviation of prebleed serum
from three animals as a negative control.

Fischer R.J., Port J.R., Holbrook M.G., Yinda K.C., Creusen M., ter Stege J., de Samber M, & Munster V.J. (2022). UV-C Light Completely Blocks Aerosol Transmission of Highly Contagious SARS-CoV-2 Variants WA1 and Delta in Hamsters. Environmental Science & Technology, 56(17), 12424-12430.

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SARS-CoV-2 Spike Protein Antibody Assay

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Serum samples were inactivated with γ-irradiation (2 mRad) and analyzed as previously described [63 ]. In brief, maxisorp plates (Nunc) were coated with 50 ng spike protein (generated in-house) per well and incubated overnight at 4°C. After blocking with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 h at room temperature (RT), serially diluted 2-fold serum samples (duplicate, in blocking buffer) were incubated for 1 h at RT. Spike-specific antibodies were detected with goat anti-hamster IgG Fc (horseradish peroxidase (HRP)-conjugated, Abcam) for 1 h at RT and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120–0047). The reaction was stopped with KPL stop solution (Seracare) and read at 450 nm. Plates were washed 3 to 5 × with PBS-T (0.1% Tween) for each wash. The threshold for positivity was calculated as the average plus 3 × the standard deviation of negative control hamster sera.

Port J.R., Yinda C.K., Owusu I.O., Holbrook M., Fischer R., Bushmaker T., Avanzato V.A., Schulz J.E., van Doremalen N., Clancy C.S, & Munster V.J. (2020). SARS-CoV-2 disease severity and transmission efficiency is increased for airborne but not fomite exposure in Syrian hamsters.. bioRxiv.

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SARS-CoV-2 Spike Protein Antibody Assay

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Serum samples were inactivated with γ-irradiation (2 mRad) and analyzed as previously described [28 (link)]. In brief, maxisorp plates (Nunc) were coated with 50 ng spike protein (generated in-house) per well and incubated overnight at 4 °C. After blocking with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 h at room temperature (RT), serially diluted 2-fold serum samples (duplicate, in blocking buffer) were incubated for 1 h at RT. Spike-specific antibodies were detected with goat anti-hamster IgG Fc (horseradish peroxidase (HRP)-conjugated, Abcam) for 1 hr at RT and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120-0047, Milford, MA, USA). The reaction was stopped with KPL stop solution (Seracare) and read at 450 nm. Plates were washed 3 to 5× with PBS-T (0.1% Tween) for each wash. The threshold for positivity was calculated as the average plus 3× the standard deviation of negative control hamster sera.

Port J.R., Adney D.R., Schwarz B., Schulz J.E., Sturdevant D.E., Smith B.J., Avanzato V.A., Holbrook M.G., Purushotham J.N., Stromberg K.A., Leighton I., Bosio C.M., Shaia C, & Munster V.J. (2021). High-Fat High-Sugar Diet-Induced Changes in the Lipid Metabolism Are Associated with Mildly Increased COVID-19 Severity and Delayed Recovery in the Syrian Hamster. Viruses, 13(12), 2506.

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SARS-CoV-2 Spike Protein Antibody ELISA

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Maxisorp plates (Nunc) were coated with 50 ng spike protein per well and incubated overnight at 4°C. After blocking with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 h at room temperature (RT), serially diluted 2-fold serum samples (duplicate, in casein) were incubated for 1 h at RT. Spike-specific antibodies were detected with goat anti-mouse IgM or IgG Fc (horseradish peroxidase (HRP)-conjugated, Abcam) for 1 h at RT and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120–0047). The reaction was stopped with KPL stop solution (Seracare) and read at 450 nm. Plates were washed 3x with PBS-T (0.1% Tween) in between steps. The threshold for positivity was calculated as the average plus 3x the standard deviation of negative control mouse sera.

Yinda C.K., Port J.R., Bushmaker T., Offei Owusu I., Purushotham J.N., Avanzato V.A., Fischer R.J., Schulz J.E., Holbrook M.G., Hebner M.J., Rosenke R., Thomas T., Marzi A., Best S.M., de Wit E., Shaia C., van Doremalen N, & Munster V.J. (2021). K18-hACE2 mice develop respiratory disease resembling severe COVID-19. PLoS Pathogens, 17(1), e1009195.

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SARS-CoV-2 Spike Protein Antibody Assay

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Serum samples were analyzed as previously described [57 (link)]. In brief, maxisorp plates (Nunc) were coated with 50 ng spike protein (generated in-house) per well. Plates were incubated overnight at 4°C. Plates were blocked with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 hour at room temperature. Serum was diluted 2-fold in blocking buffer and samples (duplicate) were incubated for 1 hour at room temperature. Secondary goat anti-hamster IgG Fc (horseradish peroxidase (HRP)-conjugated, Abcam) spike-specific antibodies were used for detection and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120–0047). The reaction was stopped with KPL stop solution (Seracare) and plates were read at 450 nm. The threshold for positivity was calculated as the average plus 3 × the standard deviation of negative control hamster sera.

Port J.R., Yinda C.K., Riopelle J.C., Weishampel Z.A., Saturday T.A., Avanzato V.A., Schulz J.E., Holbrook M.G., Barbian K., Perry-Gottschalk R., Haddock E., Martens C., Shaia C.I., Lambe T., Gilbert S.C., van Doremalen N, & Munster V.J. (2022). Infection- or vaccine mediated immunity reduces SARS-CoV-2 transmission, but increases competitiveness of Omicron in hamsters. bioRxiv.

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SARS-CoV-2 Spike Protein Antibody Assay

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Serum samples were analyzed as previously described^{55 (link)}. In brief, maxisorp plates (Nunc) were coated with 50 ng spike protein (generated in-house) per well. Plates were incubated overnight at 4°C. Plates were blocked with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 hours at room temperature (RT). Serum was diluted 2-fold in blocking buffer and samples (duplicate) were incubated for 1 hours at RT. Secondary goat anti-hamster IgG Fc (horseradish peroxidase (HRP)-conjugated, Abcam) spike-specific antibodies were used for detection and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120–0047). The reaction was stopped with KPL stop solution (Seracare) and plates were read at 450 nm. The threshold for positivity was calculated as the average plus 3 × the standard deviation of negative control hamster sera.

Port J.R., Yinda C.K., Avanzato V.A., Schulz J.E., Holbrook M.G., van Doremalen N., Shaia C., Fischer R.J, & Munster V.J. (2021). Increased aerosol transmission for B.1.1.7 (alpha variant) over lineage A variant of SARS-CoV-2. bioRxiv.

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SARS-CoV-2 Spike Protein IgG ELISA

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Serum samples were analyzed as previously described (Yinda et al., 2021 (link)). In brief, maxisorp plates (Nunc) were coated with 50 ng spike protein (generated in-house, purified recombinant) per well. Plates were incubated overnight at 4 °C. Plates were blocked with casein in phosphate buffered saline (PBS) (Thermo Fisher) for 1 hr at room temperature (RT). Serum was diluted twofold in blocking buffer and samples (duplicate) were incubated for 1 hr at RT. Secondary goat anti-hamster IgG Fc (horseradish peroxidase (HRP)-conjugated, Cat.No. 5220–0371 Lot. 10492253, Seracare) antibodies were used for detection and KPL TMB 2-component peroxidase substrate kit (SeraCare, Cat.No. 5120–0047) was used for visualization. The reaction was stopped with KPL stop solution (Seracare) and plates were read at 450 nm. The threshold for positivity was calculated as the average plus 3 x the standard deviation of negative control hamster sera.

Port J.R., Morris D.H., Riopelle J.C., Yinda C.K., Avanzato V.A., Holbrook M.G., Bushmaker T., Schulz J.E., Saturday T.A., Barbian K., Russell C.A., Perry-Gottschalk R., Shaia C., Martens C., Lloyd-Smith J.O., Fischer R.J, & Munster V.J. (2024). Host and viral determinants of airborne transmission of SARS-CoV-2 in the Syrian hamster. eLife, 12, RP87094.

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SARS-CoV-2 Spike Protein Antibody ELISA

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Yinda C.K., Port J.R., Bushmaker T., Owusu I.O., Avanzato V.A., Fischer R.J., Schulz J.E., Holbrook M.G., Hebner M.J., Rosenke R., Thomas T., Marzi A., Best S.M., de Wit E., Shaia C., van Doremalen N, & Munster V.J. (2020). K18-hACE2 mice develop respiratory disease resembling severe COVID-19. bioRxiv.

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SARS-CoV-2 Spike Protein Antibody Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

Serum samples were inactivated with γ-irradiation (2 mRad) and analyzed as previously described (Yinda et al., 2020 ). In brief, maxisorp plates (Nunc) were coated with 50 ng spike protein (generated in-house) per well and incubated overnight at 4 °C. After blocking with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 h at room temperature (RT), serially diluted 2-fold serum samples (duplicate, in blocking buffer) were incubated for 1 h at RT. Spike-specific antibodies were detected with goat anti-hamster IgG Fc (horseradish peroxidase (HRP)-conjugated, Abcam) for 1 h at RT and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120–0047). The reaction was stopped with KPL stop solution (Seracare) and read at 450 nm. Plates were washed 3 to 5 × with PBS-T (0.1 % Tween) for each wash. The threshold for positivity was calculated as the average plus 3 × the standard deviation of negative control hamster sera.

Port J.R., Adney D.R., Schwarz B., Schulz J.E., Sturdevant D.E., Smith B.J., Avanzato V.A., Holbrook M.G., Purushotham J.N., Stromberg K.A., Leighton I., Bosio C.M., Shaia C, & Munster V.J. (2021). Western diet increases COVID-19 disease severity in the Syrian hamster. bioRxiv.

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Spike Protein Antibody ELISA

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Serum samples were analyzed as previously described^{50 (link)}. In brief, maxisorp plates (Nunc) were coated with 50 ng Lineage A spike protein (generated in-house) per well. Plates were incubated overnight at 4 °C. Plates were blocked with casein in phosphate buffered saline (PBS) (ThermoFisher) for 1 h at room temperature. Serum was diluted 2-fold in blocking buffer and samples (duplicate) were incubated for 1 h at room temperature. Secondary goat anti-hamster IgG Fc (Cat.No. 5220-0371 Lot. 10492253, Seracare; each lot is tested to assure specificity and lot-to-lot consistency using an in-house ELISA assay. Reference number: 14-22-06 (https://www.seracare.com/AntiHamster-IgG-HL-Antibody-PeroxidaseLabeled-5220-0371/)) spike-specific antibodies were used (diluted at 2500X) for detection and visualized with KPL TMB 2-component peroxidase substrate kit (SeraCare, 5120-0047). The reaction was stopped with KPL stop solution (Seracare) and plates were read at 450 nm. The threshold for positivity was calculated as the average plus 3 x the standard deviation of negative control hamster sera. Endpoint titers were determined.

Port J.R., Yinda C.K., Riopelle J.C., Weishampel Z.A., Saturday T.A., Avanzato V.A., Schulz J.E., Holbrook M.G., Barbian K., Perry-Gottschalk R., Haddock E., Martens C., Shaia C.I., Lambe T., Gilbert S.C., van Doremalen N, & Munster V.J. (2023). Infection- or AZD1222 vaccine-mediated immunity reduces SARS-CoV-2 transmission but increases Omicron competitiveness in hamsters. Nature Communications, 14, 6592.

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