For detecting NP-specific antibodies in sera, wells were coated with 1 μg/mL NP23-BSA or NP2-BSA in coating buffer (Bicarbonate-carbonate buffer, pH 9.6) overnight. Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37°C for 1 h, washed twice, and incubated with indicated sera samples at 37°C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37°C for 1 h after washing with a buffer for four times. The reaction was further developed with tetramethylbenzidine (TMB), then stopped by 2N H2SO4, and read at 450 nm. Sera from influenza-challenged mice were assayed using total viral lysate prepared from stocks of strain Influenza A/PR8/34.
Anti mouse igg3
Manufactured by Southern Biotech
Sourced in United States
Anti-mouse IgG3 is a laboratory reagent used for the detection and quantification of mouse IgG3 antibodies in various immunoassays. It is a highly specific and sensitive tool for researchers studying mouse immunology and antibody responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg2b, by Southern Biotech (7 mentions)
Anti mouse igg2c, by Southern Biotech (5 mentions)
Blocking protein, by Bio-Rad (2 mentions)
Anti mouse igg2a, by Southern Biotech (2 mentions)
Anti mouse ige, by Southern Biotech (2 mentions)
Hrp conjugated goat anti mouse igg, by Southern Biotech (2 mentions)
Anti mouse iga, by Southern Biotech (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Anti mouse iga, by Fortis Life Sciences (1 mentions)
Epoch elisa reader, by Agilent Technologies (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Casein, by Merck Group (1 mentions)
96 well microtiter plate, by Corning (1 mentions)
Multiskan spectrum 1500, by Thermo Fisher Scientific (1 mentions)
Calf thymus dsdna, by Merck Group (1 mentions)
1 step turbo tmb elisa, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti mouse igg3
1
Detecting NP-specific Antibodies in Sera
2
Quantification of Antigen-Specific Immunoglobulins
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To determine the amount of antigen-specific immunoglobulins in serum, purified RBD without SpyCatcher fusion region, or purified AP205-SpyTag was used to coat the plate. Serial diluted sera were incubated with the plate, and the horseradish peroxidase (HRP)-conjugated anti-mouse IgA, anti-mouse IgG (Bethyl Laboratories, USA), anti-mouse IgM, anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2a/c, anti-mouse IgG3 (Southern Biotech, USA), or anti-monkey IgG (Abcam, UK) was used for detection. The 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich) was used as the HRP substrate, and the optical density at 450nm was measured by a microplate reader (SpectraMax, Molecular Devices, USA). Antibody titers were determined as the reciprocal of the highest dilution that gave an optical density value that was above ten times of the standard deviation value measured from the serum-free wells.
3
OVA-specific Antibody Titers in Mice
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Ten days after tumor injection, mice were bled by intracardiac puncture upon euthanasia. The blood was collected in EDTA-containing tubes, spun down at 1000 xg for 5 min and the plasma was collected and stored at −80°C until analysis. ELISA plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with 10 μg/mL OVA (Sigma-Aldrich, St. Louis, MO, USA) in PBS overnight at 4°C, and blocked with casein (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at room temperature. The plates were washed with PBST, and plasma diluted in casein was added to the wells, starting at a concentration of 1:100 and serially diluted by 10, for 2 h at room temperature. The plates were washed again, and the following HRP-conjugated antibodies were used for detection: anti-mouse IgG1 (#1070-05), anti-mouse IgG2a (#1080-05), anti-mouse IgG2b (#1090-05) and anti-mouse IgG3 (#1100-05) from Southern Biotech (Birmingham, AL, USA). The plates were revealed with TMB substrate (EMD Millipore) and stopped with 2N H2SO4. Absorbance at 450 nm was read using an Epoch ELISA reader (BioTek, Winooski, VT, USA), and corrected by the absorbance at 570 nm. Antibody titers were determined as the highest plasma dilution for which the corrected absorbance was twice the background level. The area under curve (AUC) was calculated as area under the titration curve of the log10(corrected absorbance over background).
4
Detecting NP-specific Antibodies in Sera
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For detecting NP-specific antibodies in sera, wells were coated with 1 μg/mL NP23-BSA or NP2-BSA in coating buffer (Bicarbonate-carbonate buffer, pH 9.6) overnight. Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37°C for 1 h, washed twice, and incubated with indicated sera samples at 37°C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37°C for 1 h after washing with a buffer for four times. The reaction was further developed with tetramethylbenzidine (TMB), then stopped by 2N H2SO4, and read at 450 nm. Sera from influenza-challenged mice were assayed using total viral lysate prepared from stocks of strain Influenza A/PR8/34.
5
Measuring Cytokine and Autoantibody Levels
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TNF-α and MCP-1 levels were measured using ELISA kits (R&D Systems, USA). The blood serum and cell supernatant were diluted and studied using a standard curve. All of the samples were measured in triplicate. The procedure was performed according to the manufacturer's instructions.
Anti-dsDNA antibody levels in the serum were determined by ELISA, as described previously (32 (link)). Briefly, 96-well microtiter plates (Costar) were pretreated with calf thymus dsDNA (Sigma-Aldrich) for 2 h at 37°C and then placed overnight at 4°C. After being washed with PBS containing 0.05% Tween-20 (PBST), the plates were blocked with 1% BSA for 1 h. After the plates were incubated with a 1:100 dilution of mouse serum, the levels of anti-dsDNA antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG2c, anti-mouse IgG3, anti-mouse IgM, anti-mouse IgA, and anti-mouse IgE (all from Southern Biotech). Tetramethylbenzidine (TMB) substrate was used for the development, and absorbance at 450 nm was measured on a Thermo Multiskan Spectrum 1500.
Anti-dsDNA antibody levels in the serum were determined by ELISA, as described previously (32 (link)). Briefly, 96-well microtiter plates (Costar) were pretreated with calf thymus dsDNA (Sigma-Aldrich) for 2 h at 37°C and then placed overnight at 4°C. After being washed with PBS containing 0.05% Tween-20 (PBST), the plates were blocked with 1% BSA for 1 h. After the plates were incubated with a 1:100 dilution of mouse serum, the levels of anti-dsDNA antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG2c, anti-mouse IgG3, anti-mouse IgM, anti-mouse IgA, and anti-mouse IgE (all from Southern Biotech). Tetramethylbenzidine (TMB) substrate was used for the development, and absorbance at 450 nm was measured on a Thermo Multiskan Spectrum 1500.
6
Quantifying Mouse Antibody Titers via ELISA
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Micro-vinyl plates (96-well, Corning Costar, Corning, NY, USA) were coated with a standard curve of purified IgG1 (BD Pharminogen #554121, San Diego, CA, USA), IgG2b (eBioscience eBMG2b #14732-82, San Diego, CA, UAS), IgG2c (GeneTex #GTX35043, Irvine, CA, USA), or IgG3 (eBioscience #14-4742-82, San Diego, CA, USA) protein while remaining wells were coated with 0.5 μg/mL BaL gp120 recombinant protein (NIH AIDS reagent #4961, Germantown, MD, USA) in a sodium bicarbonate buffer and incubated overnight. Plates were washed with 0.05% PBST 3 times and blocked with 5% BSA in PBS. Mouse serum was diluted in PBS at 1:500 for IgG2b and IgG3 and 1:1000 for IgG1 and IgG2c, and incubated overnight at 4 °C. The following day, plates were washed 5 times with 0.05% PBST and one of the following secondary goat anti-mouse HRP antibodies were added for 2 h: anti-Mouse IgG1 (Southern Biotech 1070-05, Birmingham, AL, USA), anti-Mouse IgG2b (Southern Biotech 1090-05, Birmingham, AL, USA), anti-Mouse IgG2c (Southern Biotech 1079-05, Birmingham, AL, USA), or anti-Mouse IgG3 (Southern Biotech 1100-05, Birmingham, AL, USA). After washing 6 times with 0.05% PBST, 1-Step™ Turbo TMB-ELISA (Thermo Scientific #34022, Rockford, IL, USA) reagent was added and incubated for 20 min at room temperature. Sulfuric acid was used to stop the reaction. Plates were analyzed on a spectrophotometer at 450 nm.
7
ELISA for Ig Quantification in Supernatants
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