Serum antibody responses were analyzed by ELISA. High-binding plates (Greiner Bio-One, Germany) were coated overnight at 4 °C with 100 µL of 5 µg mL−1 of soluble proteins, p*17 C and K4S2C, diluted in TBS buffer. The plates were blocked with 200 µL of 3% BSA in TBS buffer for 1 h at 25 °C. After three times washes with TBST, 100 µL of serially diluted mice serum samples (from 1:200 to 1:25, 600) was used as primary polyclonal antibodies and incubated for 1 h at 25 °C. Plates were washed three times with TBST before the incubation with the secondary antibodies, goat-anti-mouse IgG-HRP, goat-anti-mouse IgG1-HRP, goat-anti-mouse IgG2a-HRP, goat-anti-mouse IgG2b-HRP, and goat-anti-mouse IgG3-HRP (Abcam, United Kingdom), diluted 1:20,000 with TBST at 25 °C for 1 h. After washing three times with TBST, 100 μL of OPD (Sigma-Aldrich, USA) was added on plates and incubated for 15 min at 25 °C and the reaction was stopped using 50 µL of 0.5 N H2SO4. The results were measured at 490 nm using Elx808iu ultramicrotiter plate reader (Bio-Tek Instruments Inc., USA).
Goat anti mouse igg2b hrp
Manufactured by Abcam
Sourced in United Kingdom
Goat-anti-mouse IgG2b-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse IgG2b antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg2a hrp, by Abcam (3 mentions)
Goat anti mouse igg1 hrp, by Abcam (2 mentions)
Goat anti mouse igg hrp, by Abcam (1 mentions)
High binding plates, by Greiner (1 mentions)
Elx808iu ultramicrotiter plate reader, by Agilent Technologies (1 mentions)
Sigmafast opd tablet, by Merck Group (1 mentions)
Opteia mouse ige elisa kit, by BD (1 mentions)
Immulon 4hbx 96 well plates, by Thermo Fisher Scientific (1 mentions)
Fitc anti mouse cd19, by BioLegend (1 mentions)
Goat anti mouse igg3, by Abcam (1 mentions)
Goat anti mouse igg horseradish peroxidase hrp, by Merck Group (1 mentions)
3 protocols using goat anti mouse igg2b hrp
1
Serum Antibody Response Profiling by ELISA
2
Anti-rhGAA Antibody Quantification Assays
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Anti-rhGAA IgG1, IgG2a, IgG2b and IgM assays were developed and optimized. Immulon 4HBX 96-well plates (Thermo-Scientific) were coated with rhGAA protein and incubated overnight at 4οC. The following standards- IgG1κ (4000 ng/mL–62.5 ng/mL), IgG2a and IgG2b (1000 ng/mL–15 ng/mL), IgM (400 ng/mL – 6.25 ng/mL) were coated overnight at 4οC at 2-fold dilutions. Experimental mouse plasma at a 1:50 dilution was used for IgG1, IgG2a and IgG2b ELISA while 1:20 dilution was used for IgM ELISA. Plasma samples were incubated for 2 hours at room temperature. The secondary detection antibodies rat anti-mouse IgG1 heavy chain-HRP (AbD Serotec, UK), goat anti-mouse IgG2a- HRP (Abcam, MA), goat anti-mouse IgG2b-HRP (Abcam, MA) or rat anti-mouse IgM-HRP (SouthernBiotech, AL) were incubated for 2 hours at 37οC. Plates were allowed to develop for 5 to10 minutes in a solution containing Sigmafast OPD tablets (Sigma, MO) for color production. Plates were washed three times between procedures with tris wash buffer. Total IgE was measured using OptEIA mouse IgE ELISA Kit (BD Bioscience, CA). A colorimetric plate reader (BD Biosciences, San Jose, CA) was used to read the 96-well clear ELISA plates.
3
Antibody Sources for Murine Immunoassays
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Antibodies used in ELISAs for IgG, IgG subclasses, and IgE were purchased as follows: goat anti-mouse IgG-horseradish peroxidase (HRP) was purchased from Sigma-Aldrich (St Louis, Mo), and the following antibodies were all purchased from Abcam (Cambridge, United Kingdom): goat anti-mouse IgE-HRP, goat anti-mouse IgG 1 -HRP, goat anti-mouse IgG 2a -HRP, goat anti-mouse IgG 2b -HRP, and goat anti-mouse IgG 3 . Antibodies for flow cytometric analyses were purchased from BioLegend (San Diego, Calif), as follows: anti-mouse CD45 in fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin, and peridinin-chlorophyllprotein complex/Cy5.5 and anti-mouse CD19-FITC, anti-mouse CD124-PE, and mouse isotype controls in FITC, PE, allophycocyanin, and peridinin-chlorophyll-protein complex/Cy5.5. Antibody against serotype 19 pneumococcal strains (Rb anti-S pneumoniae serotype 19) was provided by Dr Moon Nahm and Dr Brady Spencer (University of Alabama at Birmingham, Birmingham, Ala) for this work. Total IgE levels were examined by using a specific kit for Mouse IgE (BioLegend), according to the manufacturer's instructions. M pneumoniae P1-adhesin carboxy terminus (recombinant P1 [rP1]) peptide was purchased from ProSpec Biosciences (Ness-Ziona, Israel). Grade V ovalbumin (OVA) was purchased from Sigma-Aldrich for immunization, airway challenge, and enzyme immunoassay.
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