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1xabts development solution

Manufactured by Thermo Fisher Scientific
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1xABTS development solution is a colorimetric reagent used in various analytical applications. It is designed to facilitate the detection and quantification of enzyme-labeled molecules in assays such as enzyme-linked immunosorbent assays (ELISAs). The solution provides a substrate for enzymatic reactions, resulting in a colored product that can be measured spectrophotometrically.

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5 protocols using 1xabts development solution

1

Serum ELISA for IgG Antibody Detection

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Serum ELISAs were performed by coating Corning 96-well clear flat bottom high bind microplates with 100 μL of protein at 5 μg/mL in PBS. Plates were incubated overnight at 4°C. Coating solution was removed, and plates were blocked using 1% BSA in PBS with 1% Tween for 60 minutes at room temperature. Blocking solution was removed. Sera were diluted 1:40 in PBS, and 5-fold serial dilution was performed. CR3022 IgG at a starting dilution of 5 μg/mL with 5-fold serial dilution was used as a positive control. 40 μL of primary antibody solution was applied to each well. Primary incubation occurred for 90 minutes at room temperature. Plates were then washed three times with PBS-Tween. HRP-conjugated rabbit anti-mouse IgG antibody (Abcam) at a concentration of 1:20,000 in PBS and a volume of 150 μL was used as a secondary antibody. Secondary incubation occurred for 60 minutes at room temperature. Plates were then washed three times with PBS-Tween. 1xABTS development solution (ThermoFisher) was applied as outlined in the manufacturer’s recommendations. Development was stopped after 30 minutes with a 1% SDS solution. Plates were read at 405 nm using a SectraMax iD3 plate reader (Molecular Devices).
Hauser B.M., Sangesland M., Lam E.C., Feldman J., Balazs A.B., Lingwood D, & Schmidt A.G. (2022). Humoral responses to the SARS-CoV-2 spike and receptor binding domain in context of pre-existing immunity confer broad sarbecovirus neutralization. Frontiers in Immunology, 13, 902260.
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2

ELISA Antibody Quantification Protocol

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Serum ELISAs were executed using 96-well, clear, flat-bottom, high bind microplates (Corning). These plates were coated with 100 μL of protein, which were adjusted to a concentration of 5 μg/mL (in PBS). Plates were incubated overnight at 4°C. After incubation, plates had their coating solution removed and were blocked using 1% BSA in PBS with 1% Tween. This was done for 60 minutes at room temperature. This blocking solution was removed, and sera was diluted 40-fold in PBS. A 5-fold serial dilution was then performed. CR3022 IgG, similarly serially diluted (5-fold) from a 5 μg/mL starting concentration, was used as a positive control. 40 μL of primary antibody solution was used per well. Following this, samples were incubated for 90 minutes at room temperature. Plates were washed three times using PBS-Tween. 150 μL of HRP-conjugated rabbit anti-mouse IgG antibody, sourced commercially from Abcam (at a 1:20,000 dilution in PBS), was used for the secondary incubation. Secondary incubation was performed for one hour, similarly at room temperature. Plates were subsequently washed three times using PBS-Tween. 1xABTS development solution (ThermoFisher) was used according to the manufacturer’s protocol. Development was abrogated after 30 minutes using a 1% SDS solution, and plates were read using a SectraMaxiD3 plate reader (Molecular Devices) for absorbance at 405 nm.
Hauser B.M., Sangesland M., St. Denis K.J., Lam E.C., Case J.B., Windsor I.W., Feldman J., Caradonna T.M., Kannegieter T., Diamond M.S., Balazs A.B., Lingwood D, & Schmidt A.G. (2022). Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting. Cell Reports, 38(12), 110561.
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3

Serum ELISA for Antibody Detection

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Serum ELISAs were performed using 96-well, clear, flat-bottom, high bind microplates (Corning). These plates were coated with 100 mL of protein, which were adjusted to a concentration of 5 mg/mL (in PBS). Plates were incubated overnight at 4°C. After incubation, plates had their coating solution removed and were blocked using 1% BSA in PBS with 1% Tween. This was done for 60 min nutating at room temperature. This blocking solution was removed, and sera was diluted 40-fold in PBS. A 4-fold serial dilution was then performed. CH67 IgG, similarly serially diluted (4-fold) from a 5 mg/mL starting concentration, was used as a positive control. 40 μL of primary antibody solution was used per well. Following this, samples were incubated for 90 min at room temperature. Plates were rinsed three times using PBS-Tween. 150 μL of HRP-conjugated rabbit anti-mouse IgG antibody, sourced commercially from Abcam (1:20,000 dilution in PBS), was used for the secondary incubation, performed for one hour, similarly nutating at room temperature. Plates were then rinsed three times using PBS-Tween. 150 μL of 1xABTS development solution (ThermoFisher) was used according to the manufacturer’s protocol, and stopped after 30 min using 100 μL of a 1% SDS solution, and plates were read using a SectraMaxiD3 plate reader (Molecular Devices) at 405 nm.
Caradonna T.M., Ronsard L., Yousif A.S., Windsor I.W., Hecht R., Bracamonte-Moreno T., Roffler A.A., Maron M.J., Maurer D.P., Feldman J., Marchiori E., Barnes R.M., Rohrer D., Lonberg N., Oguin TH I.I.I., Sempowski G.D., Kepler T.B., Kuraoka M., Lingwood D, & Schmidt A.G. (2022). An epitope-enriched immunogen expands responses to a conserved viral site. Cell reports, 41(6), 111628.
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4

Serum ELISA for Detection of Protein Antibodies

Cited 1 time
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Serum ELISAs were performed as previously described [14 (link)]. Briefly, plates were coated with 100 μL of protein at 5 μg/mL overnight at 4 °C, then blocked with 150 μL of 1% BSA in PBS-Tween for 1 hour at room temperature. Sera was added at a starting dilution of 1:40 with a serial 5-fold dilution and incubated for 90 minutes at room temperature. Plates were washed, and 150 μL of HRP-conjugated anti-mouse IgG (Abcam) was added at 1:20,000. After a 1-hour secondary incubation at room temperature, plates were washed and 150 μL of 1xABTS development solution (ThermoFisher) was added. Plates were developed for 30 minutes at room temperature before stopping with 100 μL of 1% SDS to read on a SpectraMaxiD3 plate reader (Molecular Devices) for absorbance at 405 nm.
Hauser B.M., Sangesland M., Lam E.C., Denis K.J., Sheehan M.L., Vu M.L., Cheng A.H., Balazs A.B., Lingwood D, & Schmidt A.G. (2023). Heterologous sarbecovirus receptor binding domains as scaffolds for SARS-CoV-2 receptor binding motif presentation. bioRxiv.
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5

Serum ELISA for Antibody Detection

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Serum ELISAs were performed using 96-well, clear, flat-bottom, high bind microplates (Corning). These plates were coated with 100 mL of protein, which were adjusted to a concentration of 5 mg/mL (in PBS). Plates were incubated overnight at 4°C. After incubation, plates had their coating solution removed and were blocked using 1% BSA in PBS with 1% Tween. This was done for 60 min nutating at room temperature. This blocking solution was removed, and sera was diluted 40-fold in PBS. A 4-fold serial dilution was then performed. CH67 IgG, similarly serially diluted (4-fold) from a 5 mg/mL starting concentration, was used as a positive control. 40 μL of primary antibody solution was used per well. Following this, samples were incubated for 90 min at room temperature. Plates were rinsed three times using PBS-Tween. 150 μL of HRP-conjugated rabbit anti-mouse IgG antibody, sourced commercially from Abcam (1:20,000 dilution in PBS), was used for the secondary incubation, performed for one hour, similarly nutating at room temperature. Plates were then rinsed three times using PBS-Tween. 150 μL of 1xABTS development solution (ThermoFisher) was used according to the manufacturer’s protocol, and stopped after 30 min using 100 μL of a 1% SDS solution, and plates were read using a SectraMaxiD3 plate reader (Molecular Devices) at 405 nm.
Caradonna T.M., Ronsard L., Yousif A.S., Windsor I.W., Hecht R., Bracamonte-Moreno T., Roffler A.A., Maron M.J., Maurer D.P., Feldman J., Marchiori E., Barnes R.M., Rohrer D., Lonberg N., Oguin TH I.I.I., Sempowski G.D., Kepler T.B., Kuraoka M., Lingwood D, & Schmidt A.G. (2022). An epitope-enriched immunogen expands responses to a conserved viral site. Cell reports, 41(6), 111628.
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