High protein binding microtiter plates

Manufactured by Corning

High-protein binding microtiter plates provide a high-affinity surface for the immobilization of proteins. These plates are designed to maximize the binding of proteins, allowing for efficient capture and detection in various applications.

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Lab products found in correlation

Super aquablue elisa substrate, by Thermo Fisher Scientific (12 mentions) Microplate spectrophotometer, by Bio-Rad (7 mentions) Horseradish peroxidase hrp conjugated goat anti human igg antibody, by Jackson ImmunoResearch (4 mentions) Human insulin, by Merck Group (3 mentions) Calf thymus dsdna, by Thermo Fisher Scientific (3 mentions) Salmonella enterica serovar typhimurium flagellin, by Thermo Fisher Scientific (3 mentions) Keyhole limpet hemocyanin (klh), by Thermo Fisher Scientific (3 mentions) Escherichia coli lps, by Merck Group (3 mentions) Goat anti human igg hrp, by Jackson ImmunoResearch (3 mentions) Microplate spectrophotometer, by Agilent Technologies (3 mentions) Hrp conjugated goat anti human igg antibody, by Jackson ImmunoResearch (1 mentions) Prism 9, by GraphPad (1 mentions)

13 protocols using high protein binding microtiter plates

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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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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SARS-CoV-2 Spike Protein 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 the RBD) at 2 μg/ml in a 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% fetal bovine serum (FBS) for 1 h at 37°C. mAbs were serially diluted 1:3 starting at 10 μg/ml and incubated for 1 h 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 1:1,000 for 1 h at 37°C, and plates were subsequently developed with the Super AquaBlue enzyme-linked immunosorbent assay (ELISA) substrate (eBioscience). The 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 (optical density at 405 nm) units. All mAbs were tested in duplicate, and each experiment was performed twice.

Changrob S., Fu Y., Guthmiller J.J., Halfmann P.J., Li L., Stamper C.T., Dugan H.L., Accola M., Rehrauer W., Zheng N.Y., Huang M., Wang J., Erickson S.A., Utset H.A., Graves H.M., Amanat F., Sather D.N., Krammer F., Kawaoka Y, & Wilson P.C. (2021). Cross-Neutralization of Emerging SARS-CoV-2 Variants of Concern by Antibodies Targeting Distinct Epitopes on Spike. mBio, 12(6), e02975-21.

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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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Antibody Titer Quantification by ELISA

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High protein-binding microtiter plates (Costar) were coated with ovalbumin at 2 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 h at 37 °C. Sera were then serially diluted 1:50 and serially diluted 3-fold. Diluted sera were transferred to blocked ELISA plate and incubated for 1.5 h at 37 °C. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1, 2b, 2c, 3) antibody diluted 1:1000 (Jackson ImmunoResearch) was added to washed plates and were subsequently developed with Super Aquablue ELISA substrate (Invitrogen). Absorbance was measured at 405 nm on a microplate spectrophotometer (BioRad). End point titers were extrapolated from sigmoidal 4PL (where X is log concentration) standard curve for each sample using Graphpad Prism (v10). The threshold to calculate the end point titers was the mean plus 2 standard deviations of naïve mouse sera on the ELISA plate as a given sample.

Doan T.A., Forward T.S., Schafer J.B., Lucas E.D., Fleming I., Uecker-Martin A., Ayala E., Guthmiller J.J., Hesselberth J.R., Morrison T.E, & Tamburini B.A. (2024). Immunization-induced antigen archiving enhances local memory CD8+ T cell responses following an unrelated viral infection. NPJ Vaccines, 9, 66.

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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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