Super aquablue elisa substrate

Manufactured by Thermo Fisher Scientific

Super Aquablue ELISA substrate is a chromogenic substrate used in enzyme-linked immunosorbent assay (ELISA) applications. It is designed to produce a blue color change upon reaction with the enzyme used in the ELISA protocol. The intensity of the color change is proportional to the amount of target analyte present in the sample.

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Lab products found in correlation

High protein binding microtiter plates, by Corning (12 mentions) Microplate spectrophotometer, by Bio-Rad (9 mentions) Human insulin, by Merck Group (4 mentions) Salmonella enterica serovar typhimurium flagellin, by Thermo Fisher Scientific (4 mentions) Keyhole limpet hemocyanin (klh), by Thermo Fisher Scientific (4 mentions) Escherichia coli lps, by Merck Group (4 mentions) Goat anti human igg hrp, by Jackson ImmunoResearch (4 mentions) Horseradish peroxidase hrp conjugated goat anti human igg antibody, by Jackson ImmunoResearch (3 mentions) Calf thymus dsdna, by Thermo Fisher Scientific (3 mentions) Microplate spectrophotometer, by Agilent Technologies (3 mentions) Hrp conjugated goat anti human igg antibody, by Jackson ImmunoResearch (2 mentions) Fetuin, by Merck Group (2 mentions) Maxisorp plate, by Thermo Fisher Scientific (2 mentions) Synergy h1 plate reader, by Agilent Technologies (2 mentions) Superblock, by Thermo Fisher Scientific (2 mentions) Prism 9, by GraphPad (1 mentions) 96 well flat bottom plate, by Thermo Fisher Scientific (1 mentions) Pna hrpo, by Merck Group (1 mentions) Peptide coating kit, by Takara Bio (1 mentions) Hrp conjugated goat anti mouse igg antibody, by Southern Biotech (1 mentions)

Close spelling variants of this product

ELISAs (35 citations) Super AquaBlue (3 citations)

30 protocols using super aquablue elisa substrate

ELISA Binding Assay for Monoclonal Antibodies

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High-protein binding microtitre plates (Costar) were coated with 10 μg ml⁻¹ calf thymus double-stranded DNA (dsDNA; Thermo Fisher Scientific), 2 μg ml⁻¹Salmonella enterica serovar Typhimurium flagellin (Invitrogen), 5 μg ml⁻¹ human insulin (Sigma-Aldrich), 10 μg ml⁻¹ KLH (Invitrogen) and 10 μg ml⁻¹Escherichia coli LPS (Sigma-Aldrich) in PBS. Plates were coated with 10 μg ml⁻¹ cardiolipin in 100% ethanol and allowed to dry overnight. Plates were washed with water and blocked with PBS, 0.05% Tween and 1 mM EDTA. mAbs were diluted 1 μg ml⁻¹ in PBS and serially diluted fourfold and added to plates for 1.5 h. Plates were washed and goat anti-human IgG-HRP (Jackson Immunoresearch) was diluted 1:2,000 in PBS, 0.05% Tween and 1 mM EDTA. Plates were washed with water and were blocked with PBS, 0.05% Tween and 1 mM EDTA for 5 min. Plates were washed again with water and were developed with Super Aquablue ELISA substrate (eBioscience) until the positive control mAb, 3H9 (ref. ^{54 (link)}), reached an A₄₅₀ of 3. All experiments were performed in duplicate and technically replicated twice.

Guthmiller J.J., Han J., Utset H.A., Li L., Lan L.Y., Henry C., Stamper C.T., McMahon M., O’Dell G., Fernández-Quintero M.L., Freyn A.W., Amanat F., Stovicek O., Gentles L., Richey S.T., de la Peña A.T., Rosado V., Dugan H.L., Zheng N.Y., Tepora M.E., Bitar D.J., Changrob S., Strohmeier S., Huang M., García-Sastre A., Liedl K.R., Bloom J.D., Nachbagauer R., Palese P., Krammer F., Coughlan L., Ward A.B, & Wilson P.C. (2021). Broadly neutralizing antibodies target a haemagglutinin anchor epitope. Nature, 602(7896), 314-320.

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SARS-CoV-2 Antibody Binding Assay

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High-protein binding microtiter plates (Costar) were coated with recombinant SARS-CoV-2 proteins at 2 μg/ml in 1X PBS overnight at 4°C. Plates were washed the next morning with 1X PBS 0.05% Tween and blocked with 1X PBS containing 20% fetal bovine serum (FBS) for 1 hour at 37°C. Antibodies were then serially diluted 1:3 starting at 10 μg/ml and incubated for 1 hour at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody diluted 1:1000 (Jackson Immuno Research) was used to detect binding of mAbs, and plates were subsequently developed with Super Aquablue ELISA substrate (eBiosciences). Absorbance was measured at 405 nm on a microplate spectrophotometer (BioRad). To standardize the assays, control antibodies with known binding characteristics were included on each plate and the plates were developed when the absorbance of the control reached 3.0 OD₄₀₅ units. All experiments were performed in duplicate 2–3 times.

Dugan H.L., Stamper C.T., Li L., Changrob S., Asby N.W., Halfmann P.J., Zheng N.Y., Huang M., Shaw D.G., Cobb M.S., Erickson S.A., Guthmiller J.J., Stovicek O., Wang J., Winkler E.S., Madariaga M.L., Shanmugarajah K., Jansen M.O., Amanat F., Stewart I., Utset H.A., Huang J., Nelson C.A., Dai Y.N., Hall P.D., Jedrzejczak R.P., Joachimiak A., Krammer F., Diamond M.S., Fremont D.H., Kawaoka Y, & Wilson P.C. (2021). Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets. Immunity, 54(6), 1290-1303.e7.

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SARS-CoV-2 Spike Protein ELISA Assay

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Recombinant SARS-CoV-2 spike/RBD proteins were coated onto high protein–binding microtiter plates (Costar) at 2 μg/mL in PBS at 50 μL/well, and kept overnight at 4°C. Plates were washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked with 150 μL of PBS containing 20% FBS for 1 hour at 37°C. Monoclonal antibodies were serially diluted 3-fold starting from 10 μg/mL in PBS and incubated in the wells for 1 hour at 37°C. Plates were then washed and incubated with HRP-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch; 109-035-098), 1:1,000) for 1 hour at 37°C. After washing, 100 μL of Super AquaBlue ELISA substrate (eBioscience) was added per well. Absorbance was measured at 405nm on a microplate spectrophotometer (Bio-Rad). The assays were standardized using control antibody S144-509 (15 (link)), with known binding characteristics in every plate, and the plates were developed until the absorbance of the control reached an OD of 3.0. All mAbs were tested in duplicate, and each experiment was performed twice.

Changrob S., Halfmann P.J., Liu H., Torres J.L., McGrath J.J., Ozorowski G., Li L., Wilbanks G.D., Kuroda M., Maemura T., Huang M., Zheng N.Y., Turner H.L., Erickson S.A., Fu Y., Yasuhara A., Singh G., Monahan B., Mauldin J., Srivastava K., Simon V., Krammer F., Sather D.N., Ward A.B., Wilson I.A., Kawaoka Y, & Wilson P.C. (2023). Site of vulnerability on SARS-CoV-2 spike induces broadly protective antibody against antigenically distinct Omicron subvariants. The Journal of Clinical Investigation, 133(8), e166844.

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Quantitative Antibody Binding Assay

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High protein-binding microtiter plates (Costar) were coated with 8 hemagglutination units (HAU) of virus in carbonate buffer or with recombinant HA at 1 μg/ml in phosphate-buffered saline (PBS) overnight at 4°C. Plates were washed with PBS/0.05% Tween and blocked with PBS containing 20% fetal bovine serum (FBS) for 1 hour at 37°C. Antibodies were then serially diluted 1:3 starting at 10 μg/ml and incubated for 1.5 hour at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody diluted 1:1000 (Jackson ImmunoResearch) was used to detect binding of mAbs, and plates were subsequently developed with Super Aquablue ELISA substrate (eBiosciences). Absorbance was measured at 405 nm on a microplate spectrophotometer (BioRad). To standardize the assays, control antibodies with known binding characteristics were included on each plate, and the plates were developed when the absorbance of the control reached 3.0 optical density (OD) units. All experiments were performed in duplicate and replicated 2–3 times. Affinity measurements, as represented as K_d at a molar concentration (M), were calculated using Prism 9 (Graphpad) by performing a non-linear regression. Area under the curve (AUC) values were calculated using Prism 9 (Graphpad).

Guthmiller J.J., Han J., Li L., Freyn A.W., Liu S.T., Stovicek O., Stamper C.T., Dugan H.L., Tepora M.E., Utset H.A., Bitar D.J., Hamel N.J., Changrob S., Zheng N.Y., Huang M., Krammer F., Nachbagauer R., Palese P., Ward A.B, & Wilson P.C. (2021). First exposure to the pandemic H1N1 virus induced broadly neutralizing antibodies targeting hemagglutinin head epitopes. Science translational medicine, 13(596), eabg4535.

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Fetuin-based ELISA Inhibition Assay

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ELLAs were performed as previously described (60 (link)). Briefly, flat-bottom 96-well plates (Thermo Fisher Scientific) were coated with 100 μl of fetuin (Sigma-Aldrich) at 25 μg/ml and incubated at 4°C overnight. mAbs were serially diluted twofold at a starting concentration of 300 μg/ml in Dulbecco’s PBS containing CaCl₂ (0.133 g/liter) and MgCl₂ (0.1 g/liter) with 0.05% Tween 20 and 1% BSA (DPBSTBSA) and then incubated in fetuin-coated plates with an equal volume of the desired antigen dilution in DPBSTBSA. Plates were sealed and incubated for 20 hours at 37°C. Plates were then washed six times with PBS–0.05% Tween 20, and 100 ml per well of HRP-conjugated peanut agglutinin lectin (Sigma-Aldrich) in DPBSTBSA was added at room temperature (RT) for 2 hours in the dark. Plates were washed six more times and subsequently developed with Super AquaBlue ELISA substrate (eBioscience). Absorbance was read at 405 nm on a microplate spectrophotometer (Bio-Rad). Data were analyzed using Prism software, and the 50% inhibitory concentration (IC₅₀) was determined as the concentration at which 50% of NA activity was inhibited compared to negative control (PBS). All experiments were performed in duplicate two times.

Dugan H.L., Guthmiller J.J., Arevalo P., Huang M., Chen Y.Q., Neu K.E., Henry C., Zheng N.Y., Lan L.Y., Tepora M.E., Stovicek O., Bitar D., Palm A.K., Stamper C.T., Changrob S., Utset H.A., Coughlan L., Krammer F., Cobey S, & Wilson P.C. (2020). Preexisting immunity shapes distinct antibody landscapes after influenza virus infection and vaccination in humans. Science translational medicine, 12(573), eabd3601.

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Polyreactivity ELISA Protocol for Antibody Screening

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Polyreactivity ELISAs were performed as previously described(Andrews et al., 2015 ; Bunker et al., 2017 (link); Guthmiller et al., 2020 (link)). High-protein binding microtiter plates (Costar) were coated with 10 μg/ml calf thymus dsDNA (Thermo Fisher), 2 μg/ml Salmonella enterica serovar Typhimurium flagellin (Invitrogen), 5 μg/ml human insulin (Sigma-Aldrich), 10 μg/ml KLH (Invitrogen), and 10 μg/ml Escherichia coli LPS (Sigma-Aldrich) in 1X PBS. Plates were coated with 10 μg/ml cardiolipin in 100% ethanol and allowed to dry overnight. Plates were washed with water and blocked with 1X PBS/0.05%Tween/1mM EDTA. MAbs were diluted 1 μg/ml in PBS and serially diluted 4-fold, and added to plates for 1.5 hours. Goat anti-human IgG-HRP (Jackson Immunoresearch) was diluted 1:2000 in PBS/0.05%Tween/1mM EDTA and added to plates for 1 hour. Plates were developed with Super Aquablue ELISA substrate (eBioscience) until the positive control mAb, 3H9 (Shlomchik et al., 1987 (link)), reached an OD₄₀₅ of 3. All experiments were performed in duplicate.

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Spike Protein ELISA Binding Assay

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High-protein binding microtiter plates (Costar) were coated with 50 μl of recombinant proteins (either full-length spike or RBD) at 2 μg/ml in 1×PBS solution overnight at 4°C. The plates were washed 3 times the next day with 1×PBS supplemented with 0.05% Tween 20 and blocked with 175 μl of 1×PBS containing 20% FBS for 1 hour at 37°C. MAbs were serially diluted 1:3 starting at 10 μg/ml and incubated for 1 hour at 37°C. The plates were then washed 3 times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch) diluted 1:1000 for 1 hour at 37°C, and plates were subsequently developed with Super AquaBlue ELISA substrate (eBioscience). Absorbance was measured at 405 nm on a microplate spectrophotometer (Bio-Rad). To standardize the assays, control antibodies with known binding characteristics were included on each plate and the plates were developed when the absorbance of the control reached 3.0 OD405 units. All mAbs were tested in duplicate and each experiment was performed twice.

Wilson P., Changrob S., Fu Y., Guthmiller J., Halfmann P., Li L., Stamper C., Dugan H., Accola M., Rehrauer W., Zheng N.Y., Huang M., Wang J., Erickson S., Utset H., Graves H., Amanat F., Sather D.N., Krammer F, & Kawaoka Y. (2021). Cross neutralization of emerging SARS-CoV-2 variants of concern by antibodies targeting distinct epitopes on spike. Research Square.

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SARS-CoV-2 S1 and S2 Protein ELISA

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High-protein binding microtiter plates (Costar) were coated with 2 µg/ml SARS-CoV-2 S1, and S2 recombinant protein in PBS overnight at 4°C, respectively. After blocking with 3% BSA in 1 × PBS, serially diluted (1:3 ratio) mAbs starting at 10 µg/ml were added to the plates and incubated for 1 h at 37°C. Plates were washed six times with PBST and then incubated with HRP (horse radish peroxidase) conjugated goat anti-human IgG (or IgA and IgM) (JACKON). The plate was developed with Super Aquablue ELISA substrate (eBiosciences). Absorbance was measured at 405 nm on a microplate spectrophotometer (BioTek).

He B., Liu S., Xu M., Hu Y., Lv K., Wang Y., Ma Y., Zhai Y., Yue X., Liu L., Lu H., Zhou S., Li P., Mai G., Huang X., Li C., Chen S., Ye S., Zhao P., Yang Y., Li X., Jie Y., Shi M., Yang J., Shu Y, & Chen Y.Q. (2022). Comparative global B cell receptor repertoire difference induced by SARS-CoV-2 infection or vaccination via single-cell V(D)J sequencing. Emerging Microbes & Infections, 11(1), 2007-2020.

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ACE2 Binding Affinity ELISA Protocol

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ELISA was performed as described previously [73 (link)]. High-protein binding microtiter plates (Costar) were coated with 2 μg/ml human ACE2 protein in PBS overnight at 4°C respectively. After 3% BSA in PBS blocking, serially diluted S proteins 1:3 starting at 50 ng/μl were incubated for 1h at 37°C. After washing 6 times with PBST, a S2 antibody from our lab, I24, was incubated at 10 μg/ml at 37°C for 1h, after washing again, the HPR-conjugated goat anti-human IgG antibody (Jackson Immuno Research, 1:2000) was incubated for another 1h at 37°C. The plate was developed with Super Aquablue ELISA substrate (eBiosciences). Absorbance was measured at 405 nm on a microplate spectrophotometer (BioTek).

Ma Y., Li P., Hu Y., Qiu T., Wang L., Lu H., Lv K., Xu M., Zhuang J., Liu X., He S., He B., Liu S., Liu L., Wang Y., Yue X., Zhai Y., Luo W., Mai H., Kuang Y., Chen S., Ye F., Zhou N., Zhao W., Chen J., Chen S., Xiong X., Shi M., Pan J.A, & Chen Y.Q. (2023). Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2. PLOS Pathogens, 19(5), e1011123.

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SARS-CoV-2 Antibody Binding Assay

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High-protein binding microtiter plates (Costar) were coated with recombinant SARS-CoV-2 proteins at 2 µg/ml in 1X PBS overnight at 4°C. Plates were washed the next morning with 1X PBS 0.05% Tween and blocked with 1X PBS containing 20% fetal bovine serum (FBS) for 1 hour at 37°C. Antibodies were then serially diluted 1:3 starting at 10 µg/ml and incubated for 1 hour at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody diluted 1:1000 (Jackson Immuno Research) was used to detect binding of mAbs, and plates were subsequently developed with Super Aquablue ELISA substrate (eBiosciences). Absorbance was measured at 405 nm on a microplate spectrophotometer (BioRad). To standardize the assays, control antibodies with known binding characteristics were included on each plate and the plates were developed when the absorbance of the control reached 3.0 OD₄₀₅ units. All experiments were performed in duplicate 2–3 times.

Stamper C.T., Dugan H.L., Li L., Asby N.W., Halfmann P.J., Guthmiller J.J., Zheng N.Y., Huang M., Stovicek O., Wang J., Madariaga M.L., Shanmugarajah K., Jansen M.O., Amanat F., Stewart I., Changrob S., Utset H.A., Huang J., Nelson C.A., Dai Y.N., Hall P.D., Jedrzejczak R.P., Joachimiak A., Krammer F., Fremont D.H., Kawaoka Y, & Wilson P.C. (2020). Distinct B cell subsets give rise to antigen-specific antibody responses against SARS-CoV-2. Research Square.

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