Anti-RBD antibodies in sera were detected by enzyme-linked immunosorbent assay (ELISA) as described previously (34 (link), 40 (link), 44 (link)). Briefly, high binding 96-well Maxisorp plates (Thermo Scientific) were coated overnight at 4°C with 1 µg/mL of an RBD target in PBS. Plates were rinsed three times with PBS and 0.05% Tween-20 (J62844, Alfa Aesar) then blocked for 1 hour at room temperature with 1% bovine serum albumin (BSA) in PBS (Thermo Scientific 37525). Plates were rinsed three times before addition of diluted sera or nasal washes and incubated overnight at 4°C. After rinsing three times as above, anti-mouse IgG (0107-05, SouthernBiotech), anti-mouse IgG1 (1071-05, SouthernBiotech), anti-mouse IgG2a (1081-05, SouthernBiotech), or anti-mouse IgA (1165-05, SouthernBiotech) conjugated with horseradish peroxidase (HRP) were added to plates and incubated for 1 hour at room temperature. Colorimetric signals were developed with 3, 3′, 5, 5′ - tetramethylbenzidine (TMB) (T0440, Sigma) and reactions were stopped with 2N HCl. Antibody levels were quantified via absorbance readings at 450 nm and 570 nm on a microplate reader (Cytation 5; Biotek, Winooski, VT).
Anti mouse igg2a
Manufactured by Southern Biotech
Sourced in United States
Anti-mouse IgG2a is a secondary antibody used in immunological applications to detect the presence of mouse IgG2a primary antibodies. It specifically binds to the Fc region of mouse IgG2a antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse iga, by Southern Biotech (3 mentions)
96 well microtiter plate, by Corning (2 mentions)
Anti mouse igg2b, by Southern Biotech (2 mentions)
Anti mouse igg3, by Southern Biotech (2 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Elisa plate reader, by Bio-Rad (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (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)
O phenylenediamine dihydrochloride substrate, by Merck Group (1 mentions)
Horse radish peroxidase conjugated anti mouse igg, by Southern Biotech (1 mentions)
Anti mouse ige, by Southern Biotech (1 mentions)
Anti mouse igg2c, by Southern Biotech (1 mentions)
Multiskan spectrum 1500, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse igg, by Southern Biotech (1 mentions)
Calf thymus dsdna, by Merck Group (1 mentions)
7 protocols using anti mouse igg2a
1
ELISA Protocol for Anti-RBD Antibody Detection
2
ELISA Evaluation of RBD-specific Antibodies
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The lung lavage, gut-wash and sera samples from the animals were collected at eight wpi and tested for RBD-specific IgA, IgG, IgG1 and IgG2a antibodies by an ELISA. Briefly, purified RBD antigen produced in E.coli (1 μg/mL) in 100 μL carbonate buffer was used to coat 96-well microtiter plates (Corning-Costar, Corning, NY, USA) overnight at 4 ℃. Following three washes with PBST and blocking with PBST containing 3% BSA for 2 h at 37 °C, the plates were incubated with 2-fold serial dilutions of samples in PBS containing 0.5% (w/v) BSA at 37 °C for 1 h. After three wash cycles with PBST, the plates were incubated with the following HRP-labeled goat antibodies: anti-mouse IgA (1:2000, SouthernBiotech, Birmingham, AL, USA), anti-mouse IgG (1:2000, BioWorld, St. Louis, MN, USA), anti-mouse IgG1 (1:2000, Southern Biotech, USA), and anti-mouse IgG2a (1:2,000, SouthernBiotech, Birmingham, AL, USA) at 37°C for 1 h. Subsequently, the plates were washed three times and 100 µL tetramethylbenzidine substrate was added per well; the color development was stopped by adding 50 µL/well H2SO4. Optical density values were measured at 450 nm using an ELISA plate reader (Bio-Rad, Hercules, CA, USA).
3
ZIKV VLP-Based ELISA Assay
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In the ELISA assays, VLPs mimicking the 3-D structure of the virus were used to coat the plates. These VLPs were produced at the Cell Culture Engineering Lab of COPPE/UFRJ by a stable pool of HEK293 cells constitutively expressing a prME construct designed according to the sequence of BeH819966 ZIKV strain (Genbank KU365779). VLPs were purified from serum-free cell culture supernatant by a two-step chromatography process prior to use. Briefly, ELISA plates were coated with 1 ug/mL of VLPs or 150 ng EDIII protein30 (link) in PBS per well and stored overnight at room temperature. Plates were then blocked with PBS 1% BSA for 2 h at room temperature. Serum samples were initially diluted 1:40 and serially threefold diluted to 1:1080 with PBS 1% BSA and added for 2 h at room temperature. Next, the following goat secondary antibodies conjugated with horseradish peroxidase were added to the plate for 2 h at room temperature: anti-mouse IgM (1:400), anti-mouse IgG (1:8000), anti-mouse IgG1 (1:2000) or anti-mouse IgG2a (1:2000) (SouthernBiotech, Birmingham, AL). Finally, TMB substrate solution (Life Technologies) were added and plates were read at 450 nm. Plates were washed with PBS each step.
4
Enumeration of 4-44+ and NP-specific AFCs
5
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).
6
Antibody Titration by ELISA
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ELISA plates (Immunosorp, Nunc, Denmark) were coated overnight with 1 μg/ml gp140 diluted in 0.1 M carbonate buffer at 4°C. Plates were then washed three times with PBS plus 0.05% Tween and blocked with 1% BSA for 1 h at room temperature. Subsequently, serum was serially diluted in PBS plus 0.05% Tween and incubated overnight at 4°C. After washing the plates three times with PBS plus 0.05% Tween, horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1 or anti-mouse-IgG2a (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h at room temperature. After five washes with PBS plus 0.05% Tween, the o-phenylenediamine dihydrochloride substrate (Sigma-Aldrich) was added for detection of antibodies. After 20 min, the reaction was stopped with 1 M HCl and the optical density (OD) at 490 nm was read using and ELISA reader. For calculation of endpoint titers, a cutoff value of OD 0.15 was used. Results are expressed as group means+SEM.
7
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.
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