Mice sera were stored at –20°C and thawed before use. We used the following capture antibodies: unlabeled goat anti-mouse IgG1 (Southern Biotech, AL, USA; 1071–01), IgG2a (Southern Biotech; 1080–01), IgG2b (Southern Biotech; 1090–01), IgG2c (Southern Biotech; 1079–01), and IgG3 (Southern Biotech; 1100–01) and rabbit anti-human IgG4 H&L (Abcam, Cambridge, UK, ab86251). These antibodies were diluted 1:100 in phosphate-buffered saline, plated, and stored at 4°C overnight. We used isotype control mouse IgG1 (Southern Biotech; 0102–01), IgG2a (Southern Biotech; 0103–01), IgG2b (Southern Biotech; 0104–01), IgG3 (Southern Biotech; 0105–01), and human IgG4 (Sigma-Aldrich, St. Louis, MO, USA; I4639) as standards. We incubated the standards and samples in 1% bovine serum albumin for 2 h. Following this, they were washed and incubated with the detection antibodies, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 (Sothern Biotech; 1070–05), IgG2a (Southern Biotech; 1080–05), IgG2b (Southern Biotech; 1090–05), and IgG3 (Southern Biotech; 1100–05) at 1:5000 dilution and HRP-conjugated monoclonal HP6023 mouse anti-human IgG4 (Abcam; ab99817) at 1:2000 dilution. Subsequently, a chromogenic reagent was added to the reaction. A turbidimetric immunoassay for the quantification of human IgG4 concentration was outsourced to Japan SRL, Inc (Kumiyama, Japan).
Igg2a
Manufactured by Southern Biotech
Sourced in United States
IgG2a is an immunoglobulin subclass that functions as an antibody. It is produced by plasma B cells and plays a role in the adaptive immune response.
Automatically generated - may contain errors
Lab products found in correlation
Igg2b, by Southern Biotech (31 mentions)
Igg2c, by Southern Biotech (11 mentions)
Sf900ii medium, by Thermo Fisher Scientific (8 mentions)
Hrp conjugated goat anti mouse igg, by Southern Biotech (5 mentions)
Tween 20, by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Balb c mice, by Nara Biotech (3 mentions)
Hep 2 cells, by American Type Culture Collection (3 mentions)
Acrylic acid, by Tianjin Damao (3 mentions)
Horseradish peroxidase conjugated streptavidin, by Vector Laboratories (2 mentions)
Costar eia ria plates, by Corning (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Mouse anti rsv f monoclonal antibody 131 2a, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ova specific igg1, by Merck Group (2 mentions)
Alkaline phosphatase conjugated goat anti mouse igg, by Southern Biotech (2 mentions)
Hrp conjugated goat anti mouse igg1, by Southern Biotech (2 mentions)
96 well microtiter plate, by Corning (2 mentions)
P nitrophenyl phosphate, by Merck Group (2 mentions)
Elisa reader, by Agilent Technologies (2 mentions)
74 protocols using igg2a
1
Quantification of Mouse and Human Immunoglobulin Isotypes
2
Quantifying Antibody Isotype Profiles
3
Quantifying Serum Immunoglobulin Levels
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Sera were analyzed by ELISA for TSLP, total IgG, IgG1, IgG2a, and IgE levels. Serum TSLP was measured using the murine TSLP Quantikine ELISA kit (R&D Systems) according to the manufacturer’s instructions. For immunoglobulin analysis, ELISA plates were coated overnight with rat anti–mouse IgG (2.5 µg ml−1, LO-MG-COC-2), IgG1 (1.25 µg ml−1, LO-MG1-13) or IgG2a (5 µg ml−1, LO-MG2a-7) antibodies (Southern Biotech). Plates were blocked with 1% BSA in PBS and incubated with serial dilutions of mouse sera, followed by anti-Ig PO HRP-conjugated secondary (1/2000, Southern Biotech). Signal was developed by incubation with 3,3′,5,5′–tetramethylbenzidine substrate (Fluka). For IgE, plates were coated overnight with 3 µg ml−1 rat anti–mouse IgE (LO-ME-3), blocked with 1% BSA in PBS, and incubated with serial dilutions of mouse sera, followed by biotinylated rat anti–mouse IgE antibody (1/2000; Southern Biotech). Signal was developed by addition of streptavidin-HRP conjugate (1/2000; Interchim). All samples were analyzed in duplicate or triplicate by measuring the absorbance at 450 nm using a microplate reader (Versamax; Molecular Devices). Concentrations were calculated using standard curves generated with known dilutions of mouse IgG, IgG2a, IgG1 (Southern Biotech), and IgE (BD).
4
SARS-CoV-2 Antibody Quantification by ELISA
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Antigen-specific IgG (Cat. No. 1015-05, Southern Biotech, AL, USA), IgA (Cat. No.1040-05, Southern Biotech, AL, USA), IgG1 (Cat. No. 1071-01, Southern Biotech, AL, USA) and IgG2a (Cat. No.1101-01, Southern Biotech, AL, USA) levels were determined by enzyme linked immunosorbent assay (ELISA) using recombinant SARS-CoV-2 S Ptn as the capture antigen. ELISA plates (Corning, MA, USA) were coated with 4 μg/mL of S Ptn in PBS overnight at 4°C. The next morning, the coating solution was discarded and a blocking solution of PBS + 5% milk (Molico) was added for 1 h. The blocking solution from the ELISA plates was discarded and blood and BALF samples diluted in blocking solution were added for at least 2 h. After this incubation the ELISA plate was washed 5 times with a washing solution consisting of PBS + 0.05% Tween 20 and then the anti-mouse IgG, IgG1, IgG2a, and IgA-HRP detection antibodies (Southern Biotech, AL, USA) were added for another hour. The plate was washed 7 more times and TMB solution (Invitrogen, MA, USA) was added. The reaction was stopped with 1 N HCl and readed at 450nm. The normalized optical density (O.D.) was made by normalizing data from 4 different experiments using the control groups for that end. The O.D. summatory (Sum) was made by summing the values from normalized O.D. as previously described (25 (link)).
5
Autoantibody Measurement by ELISA
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Anti-chromatin, anti-ssDNA, and anti-dsDNA IgM, IgG, IgG1, and IgG2a autoantibodies, were measured by ELISA, as previously described [6 (link)]. Briefly, 96 well flat bottom plates were coated with antigen and left overnight. Serum was diluted 1 in 50 and added in triplicate to the plates. Bound antibodies were detected using alkaline-phosphatase conjugated anti-IgM, -IgG, -IgG1, -IgG2a, IgG2b, IgG2c, or IgG3 secondary reagents (Southern Biotech; Birmingham, AL). Substrate (4-nitrophenyl phosphate disodium salt hexahydrate, Sigma-Aldrich, St Louis, MO, USA) was added, and the OD of each well was measured at a wavelength of 405 nm. Values were standardized from plate to plate by running known B6 and NZB controls.
6
ELISA for Anti-Chicken Collagen II Antibodies
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Anti-CII antibodies in serum were measured by sandwich ELISA (Kwaśny-Krochin et al. 2002 (link)). In brief, plates (Costar EIA/RIA plates, Corning Incorporated, USA) were coated overnight with native chicken CII (5 μg/ml). 0.05% Tween 20 (Sigma-Aldrich, Germany) in phosphate buffer was used as a washing solution. Serial dilutions of mouse serum in PBS were added to antigen coated wells and incubated for 1 h at room temperature followed by biotin-conjugated antibodies against mouse immunoglobulins: IgG (Sigma-Aldrich, Germany), IgG1 (MP Biomedicals, USA) or IgG2a (Southern Biotech, USA). Horseradish peroxidase conjugated streptavidin (Vector, USA) was used as detection reagent; o-phenylenediamine dihydrochloride and hydrogen peroxide (both Sigma-Aldrich, Germany) were used for color development. The reaction was stopped with 3-M H2SO4 (POCH, Poland). The optical density of each sample was measured at 492 nm. The antibody levels were expressed in arbitrary ELISA units calculated from anti-CII immunoglobulin titer: 1 Unit = 1/100 titer of immunoglobulin specific to CII.
7
Bead-based Multiplex Assay for Pertussis Antibodies
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Antibodies against B. pertussis antigens mutant Ptx, Prn, FHA, Fim2/3, BrkA, Vag8, and OMV were measured using a MIA. Conjugation of antigens and OMVs to magnetic beads of different bead regions (Luminex, Austin, TX, USA) was performed using the Bio-Plex Amine Coupling kit (Bio-Rad, Hercules, CA, USA) and was described previously [20 (link)]. Serum was diluted 1:100 for IgG (subclass) and 1:1000 for anti-OMV IgG in PBS containing 0.1% Tween 20 and 3% bovine serum albumin (Sigma, Zwijndrecht, The Netherlands) and added to the conjugated beads in a 1:1 ratio. Subsequently, samples were incubated with R-Phycoerythrin (RPE)-conjugated anti-mouse IgG (1:200), IgG1 (1:200), IgG2a (1:40), IgG2b (1:200), and IgG3 (1:200) (Southern Biotech, Birmingham, AL, USA). Data was acquired with a Bio-Plex 200, processed using Bio-Plex Manager software (v6.1, Bio-Rad Laboratories, Hercules, CA, USA), corrected for the background signal, and presented as fluorescence intensities (FI). The limit of detection of each analyte was set at 15% of the highest FI-value in the dataset.
8
ELISA-Based Antibody Titer Quantification
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Antibody titers against CfaEB and dmLT were determined by ELISA. Corning 3700 384-well microtiter plates were coated with 2 µg/mL of CfaEB antigen or 1 µg/mL dmLT, and blocked with PBS (pH 7.1), 0.1% Tween-20, and 1% skim milk. 12-point 3- or 4-fold serial dilutions of the serum and IW samples were then carried out. Detection antibodies included IgG1, IgG2a, total IgG, and IgA (all conjugated to horse radish peroxidase [HRP]) (Southern Biotech). The plates were incubated in the HRP substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) for 4 min, and the reaction was stopped using 1 N H2SO4. Finally, optical density (OD) readings were taken at 450 nm, with data reduction at 570 nm using an automated plate-reader (Biotek Synergy 2). The endpoint titers were determined using the dilution at OD = 0.5.
9
RSV Antibody Characterization Protocol
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HEp-2 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Grand Island, NY) with 10% fetal bovine serum (FBS, GIBCO-BRL), 2 mM glutamine, penicillin and streptomycin (GIBCO-BRL) at 37°C with 5% CO2. Mouse anti-RSV F monoclonal antibody (131-2A) was purchased from Millipore (Billerica, MA, USA). The horseradish peroxidase (HRP) conjugated goat anti-mouse immunoglobulin G (IgG), IgG1, and IgG2a were from Southern Biotech (Birmingham, AL, USA). Monophosphoryl lipid A (MPL) was purchased from Sigma-Aldrich. RSV F purified protein was prepared in a post-fusion conformation (VRC 3089 pAH RSV A2 F dF) and provided by Vaccine Research Center (VRC), National Institute of Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA. RSV A2 G protein fragment (aa131-230) was prepared as previously described [24 (link)].
10
Quantifying SseB-specific Antibody Responses
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SseB-specific IgG and IgG subtypes IgG1, IgG2a, and IgG2b were detected using indirect ELISA as described previously [35 (link)]. Briefly, 2 μg/mL rGST-SseB were regarded as coated antigen dissolved in 50 mM carbonate buffer in 96-well plates at 4 °C for 14 h. After washing and blocking, serial double dilutions (beginning at 1:100) of the serum samples were added to rGST-SseB covered 96-well plates for 2 h at 37 °C. After washing, HRP-conjugated goat anti-mouse IgG (1:10,000, Southern Biotech, Birmingham, AL, USA), IgG1 (1:3000, Southern Biotech), IgG2a (1:3000, Southern Biotech), or IgG2b (1:3000, Southern Biotech) were added for 1 h at 37 °C. After washing, 100 μL of 3,3′,5,5′-tetramethylbenzidine substrate was added to the 96-well plates for ELISA development and then stopped with 2 M H2SO4. Finally, the absorbance was measured at 450 nm.
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