The OVA-specific IgA response was determined in ELISA with OVA-coated plates, total IgA was determined in ELISA with anti-mouse IgA (Southern biotech, USA)-coated plates for both using anti-mouse IgA biotinylated goat, anti-mouse IgA (Southern biotech, USA) for detection [22] (link) following the procedures described for measuring OVA-specific IgG titers. The titers of total IgA and of OVA-specific IgA were determined as the highest dilution factors of the assay samples with twice the average readout value of the blank. For normalization, the percentage of OVA-specific IgA titer of the total IgA titer was calculated for each sample.
Anti mouse iga
Manufactured by Southern Biotech
Sourced in United States
Anti-mouse IgA is a laboratory reagent used to detect and quantify the presence of immunoglobulin A (IgA) in mouse samples. It functions as a specific antibody that binds to mouse IgA, allowing for its identification and measurement.
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Lab products found in correlation
Anti mouse igg2a, by Southern Biotech (3 mentions)
96 well microtiter plate, by Corning (2 mentions)
Anti mouse pigr, by R&D Systems (2 mentions)
Mouse pigr, by Southern Biotech (2 mentions)
Dtmb substrate, by Thermo Fisher Scientific (2 mentions)
Anti mouse ige, by Southern Biotech (2 mentions)
Anti mouse igg2b, by Southern Biotech (2 mentions)
Anti mouse igg2c, by Southern Biotech (2 mentions)
Anti mouse igg3, by Southern Biotech (2 mentions)
Hrp conjugated goat anti mouse igg, by Southern Biotech (2 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
Hrp conjugated anti mouse igg, by Southern Biotech (1 mentions)
Elisa plate, by Thermo Fisher Scientific (1 mentions)
Elisa plate reader, by Bio-Rad (1 mentions)
Hrp conjugated goat anti mouse iga, by Southern Biotech (1 mentions)
96 well flat bottom plate, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Multiskan spectrum 1500, by Thermo Fisher Scientific (1 mentions)
Calf thymus dsdna, by Merck Group (1 mentions)
14 protocols using anti mouse iga
1
Quantifying OVA-Specific IgA Response
2
ELISA Protocol for Anti-RBD Antibody Detection
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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).
3
SARS-CoV-2 Spike Antibody ELISA Assay
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Sera and BAL from different time-points were screened for humoral response against SARS-CoV-2 spike. To measure IgG and IgA antibody levels in the plasma and BAL of mice, respectively, a SARS-CoV-2-specific enzyme-linked immunosorbent assay (ELISA) was developed. Briefly, Nunc ELISA plates were coated with SARS-CoV-2 spike protein (BEI resources-NR-52396, 100 ng total/well) diluted in carbonate/bicarbonate buffer, pH 9.6, and incubated overnight at 4 °C followed by blocking with 5% skim milk to reduce background. A total of 100 μL of diluted serum (1/25) or BAL (undiluted) harvested at different time-points from immunized mice was added to the wells and incubated at 37 °C for 1 h. Post washing (PBS-TritonX 100, 0.1%), either HRP-conjugated anti-mouse IgG (1036-05, Southern Biotech, Birmingham, AL, USA) or anti-mouse IgA (1040-05, Southern Biotech) at dilutions of 1/1000 was added to the wells and incubated at 37 °C for 1 h. Post washing, 100 μL of TMB substrate solution was added and incubated for 20 min or until color developed. The addition of 1 M sulfuric acid stopped the reaction, and plates are read at 450 nm. Binding antibody end point titers (EPTs) were calculated as described previously [17 (link)].
4
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).
5
Quantification of Murine IgA, pIgR, and Cytokines
6
Quantification of Murine IgA, pIgR, and Cytokines
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For IgA and pIgR ELISA analysis, fresh feces were collected from individual mice, homogenized in sterile PBS, and centrifuged to remove bacteria and insoluble debris. Fecal samples were diluted in sterile PBS as a 2-fold serial dilution and added to 96-well plates pre-coated overnight (at 4 °C) with 1 µg/ml anti-mouse IgA (Southern Biotechnology) or 0.5 µg/ml anti-mouse pIgR (R&D Systems). Samples were incubated at room temperature for 2 h. For IgA analysis, HRP conjugated anti-IgA (Southern Biotechnology) was added for 1 h. For pIgR ELISA analysis, biotinylated mouse pIgR antibody was added for 1 h, followed by incubation with avidin-HRP for 30 min. Plates were developed using dTMB substrate according to the manufacturer’s instructions (Thermo Fisher Scientific) and analyzed at 450nm using a microplate reader.
For ELISA analysis of cytokines (IL-23p19, IL-1β and IL-6), equal numbers of wild-type and Map3k14-cKO BMDCs were stimulated with CpG or Pam3CSK4 for 48 h, and supernatants were collected for ELISA analysis using an ELISA kits (Thermo Fisher Scientific) according to the manufacturer’s instructions. Detection limits were 16 pg/ml for IL-23p19, 1.2 pg/ml for IL-1β, and 4 pg/ml for IL-6.
For ELISA analysis of cytokines (IL-23p19, IL-1β and IL-6), equal numbers of wild-type and Map3k14-cKO BMDCs were stimulated with CpG or Pam3CSK4 for 48 h, and supernatants were collected for ELISA analysis using an ELISA kits (Thermo Fisher Scientific) according to the manufacturer’s instructions. Detection limits were 16 pg/ml for IL-23p19, 1.2 pg/ml for IL-1β, and 4 pg/ml for IL-6.
7
Quantifying Mouse Immunoglobulin Isotypes
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Flat-bottom 96-well plate (Nunc) was coated with 1 mg/ml of anti-mouse IgA (Southern Biotech, 1040–01), IgM (Southern Biotech, 1020–01) or IgG (Southern Biotech, 1030–01) by incubating at room temperature for 1 hour, and then blocked with 3% BSA or 5% FCS. The diluted supernatants of B cell culture were added to the wells and allowed to react for 1 hour at room temperature. As a standard, Mouse Reference Serum (Bethyl, RS10-101) was 2-fold serially diluted. Abs bound to the plate were detected with 1 mg/ml of HRP-conjugated goat anti-mouse IgA (Southern Biotech, 1040–05), IgM (Southern Biotech, 1020–05) or IgG (Southern Biotech, 1033–05) after 1 h incubation. Then Abs bound to the plate were developed with TMB substrate (Sigma) and the reaction was terminated by sulfuric acid. Absorbance values were measured at 450 nm using plate reader (Bio-Rad).
8
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.
9
SARS-CoV-2 S RBD Antibody Detection
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S protein receptor-binding domain (S RBD)-specific antibody titers in serum and BALF were determined using ELISAs. The recombinant His-tagged SARS-CoV-2 S RBD protein (aa 319–541) was produced in Expi293F cells using a transient expression system (Thermo Fisher) according to the manufacturer's instructions and purified using Nuvia™ IMAC Resin (Bio-Rad). NUNC-MaxiSorp™ 96-well plates (Thermo Scientific) were coated with 1 μg/ml of recombinant SARS-CoV-2 S RBD protein overnight at 4 °C and blocked with 5% fetal bovine serum (FBS) in DBPS for 1 h at RT. Serially diluted samples were added to wells and incubated overnight at 4 °C. After washing four times with ELISA washing buffer (DPBS containing 0.05% Tween-20), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) or anti-mouse IgA (Southern Biotech, Birmingham, AL, USA) antibodies were used to detect S RBD-specific antibodies in the samples. Plates were incubated for 1 h at RT and washed four times with ELISA washing buffer. TMB (eBioscience, San Diego, CA, USA) was used as the substrate, and relative titers were calculated based on the absorbance at 450 nm using a SpectraMax® ABS Plus Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA).
10
ELISA for Mouse Antibody Detection
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Microplates (96-well) were coated with PT, FHA, or PRN at 3 μg/mL and incubated at 4°C overnight. Then, the plates were blocked with 3% (w/v) bovine serum albumin (BSA, Abcam) in PBS at 37°C for 2 h. Diluted serum, NW, or BALF sample was added to each microplate and incubated at 37°C for 1 h. After washing, a horseradish peroxidase (HRP)-labeled sheep anti-mouse IgG (Jackson ImmunoResearch, USA), anti-mouse IgG1 (Southern Biotech, USA), anti-IgG2a (Southern Biotech, USA), or anti-mouse IgA (Southern Biotech, USA) antibody was added to the microplate, and the plate was incubated at 37°C for 1 h. All the ELISA plates were developed using tetramethylbenzidine (TMB; Solarbio, CHN) to generate a colorimetric reaction, and the reaction was terminated with 2 mmol/L H2SO4. The absorbance of the plates at 450 nm was read. Endpoint titers were determined as the dilution that exhibited an optical density exceeding ≥2.1 times the background level (secondary antibody alone).
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