Sera collected from WT and BC-CosmcKO mice were titrated and added in duplicate into 96-well plates (Corning) precoated with polyvalent goat antibody against mouse immunoglobulins (IgM, IgG1, IgG2b, IgG2b, IgG3, IgA from Southern Biotech), and followed by HRP-conjugated goat anti-mouse IgG (Southern Biotech and Fisher Scientific) and then TMB ELISA substrate (Abcam). Absorbance was measured at 450 nm with a Multiskan Spectrum spectrophotometer (Thermo Scientific). The mouse immunoglobulin concentration was calculated from a curve constructed using mouse immunoglobulin standard as listed: IgM, IgG1, IgG2b, IgG2b, IgG3, IgA (Southern Biotech).
Igg2b
Manufactured by Southern Biotech
Sourced in United States
IgG2b is a type of immunoglobulin G (IgG) subclass that is commonly used in laboratory research and applications. It functions as an antibody, playing a role in the immune system's response to foreign substances. IgG2b has specific structural and functional characteristics that make it a valuable tool for various experimental and analytical purposes.
Automatically generated - may contain errors
Lab products found in correlation
Igg2a, by Southern Biotech (31 mentions)
Igg2c, by Southern Biotech (30 mentions)
Hrp conjugated goat anti mouse igg, by Southern Biotech (4 mentions)
P nitrophenyl phosphate, by Merck Group (3 mentions)
Sf900ii medium, by Thermo Fisher Scientific (3 mentions)
Fcr blocking mab2.4g2, by BioXCell (3 mentions)
1 2 dipalmitoyl sn glycero 3 phosphocholine, by NOF corporation (3 mentions)
N carbonyl methoxypolyethyleneglycol 2000 1 2 distearoyl sn glycero 3 phosphoethanolamine, by NOF corporation (3 mentions)
Victor3 plate reader, by PerkinElmer (2 mentions)
Igg2b, by Merck Group (2 mentions)
Hrp conjugated goat anti mouse igg1, by Southern Biotech (2 mentions)
Np conjugated ficoll, by Biosearch Technologies (2 mentions)
Hrp conjugated anti mouse igm, by Southern Biotech (2 mentions)
Biotin conjugated anti cxcr5 2g8 and fitc conjugated anti tcrβ h57 597 mabs, by BD (2 mentions)
Pe texas red conjugated streptavidin, by BD (2 mentions)
Pe cy7 conjugated anti pd 1 j43 mab, by Thermo Fisher Scientific (2 mentions)
Balb c mice, by Nara Biotech (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
4 nitrophenyl phosphate disodium salt hexahydrate, by Merck Group (1 mentions)
Biotinylated goat anti mouse igg, by Southern Biotech (1 mentions)
72 protocols using igg2b
1
Quantifying Mouse Immunoglobulin Levels
2
Quantification of Mouse and Human Immunoglobulin Isotypes
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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).
3
Quantifying Antibody Isotype Profiles
4
Quantification of Serum Ig Isotypes
5
Immunization and Infection Protocol
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For immunization, mice were injected i.p. with 50 µg NP19-KLH (Biosearch Technology) in 4mg Alum (Thermo Fisher Scientific). Blood samples were taken from the lateral tail vein on day 0, 7, 14, 21, 28 after immunization.
For infection, mice were intranasally immunized with 200pfu of Influenza A virus (PR8 strain). Blood samples were taken from the lateral tail vein on day 0 and 9 after infection.
NP- or Influenza-specific antibody titers were detected by ELISA, using NP23-BSA, NP3-BSA, or live Influenza virus for capture, and biotinylated anti–mouse IgM (Southern Biotech), IgG (Southern Biotech), IgG2b (Southern Biotech), IgG2c (Southern Biotech), and IgG3 (BD). Titers were determined from the dilution curve in the linear range of absorbance.
Total antibody levels in serum before immunization were measured by sandwich ELISA. For capture, anti-IgM II/41 (BD), anti-IgG2b (BD), and anti-IgG2c (BD) were used. Purified IgM (BD), IgG2b (Southern Biotech), or IgG2c (Bethyl) were used as standards. Biotinylated antibodies were used for detection. For IgG1, a commercial kit was used (eBioscience).
All noncommercial ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma-Aldrich) and phosphorylated nitrophenyl phosphate (Sigma-Aldrich). Absorbance at 405 nm was determined with a SPECTRAmax190 plate reader (Molecular Devices).
For infection, mice were intranasally immunized with 200pfu of Influenza A virus (PR8 strain). Blood samples were taken from the lateral tail vein on day 0 and 9 after infection.
NP- or Influenza-specific antibody titers were detected by ELISA, using NP23-BSA, NP3-BSA, or live Influenza virus for capture, and biotinylated anti–mouse IgM (Southern Biotech), IgG (Southern Biotech), IgG2b (Southern Biotech), IgG2c (Southern Biotech), and IgG3 (BD). Titers were determined from the dilution curve in the linear range of absorbance.
Total antibody levels in serum before immunization were measured by sandwich ELISA. For capture, anti-IgM II/41 (BD), anti-IgG2b (BD), and anti-IgG2c (BD) were used. Purified IgM (BD), IgG2b (Southern Biotech), or IgG2c (Bethyl) were used as standards. Biotinylated antibodies were used for detection. For IgG1, a commercial kit was used (eBioscience).
All noncommercial ELISA plates were developed with alkaline-phosphatase streptavidin (Sigma-Aldrich) and phosphorylated nitrophenyl phosphate (Sigma-Aldrich). Absorbance at 405 nm was determined with a SPECTRAmax190 plate reader (Molecular Devices).
6
Quantification of Serum Immunoglobulin Levels
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Serum Ig levels were measured by ELISA developed in hand. In brief, serum samples were incubated with ELISA plate coated with goat anti-mouse IgM, IgG1, IgG2b, IgG2c, IgG3, or IgA Abs (Southern Biotech) and detected with biotinylated goat Abs against each class (Southern Biotech) and streptavidin-conjugated alkaline phosphatase. The plate was developed by alkaline phosphatase buffer (50 mM NaHCO3, 10 mM MgCl2, pH9.8) containing phosphatase substrate tablet (S0942, Sigma-Aldrich). Purified mouse IgM, IgG1, IgG2b, IgG2c, IgG3 (Southern Biotech) were used as standard.
7
Quantification of Antigen-Specific Antibody Responses
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Ig titers were determined in culture supernatants and mouse sera using unlabeled and alkaline phosphatase–conjugated goat anti–mouse Abs (IgM, IgG1, IgG3, IgG2b, Igκ, Igλ, and total IgG; Southern Biotech) as described (Pinaud et al., 2001 (link); Sirac et al., 2006 (link)). Total Ig amounts were revealed by the addition of p-nitrophenyl phosphate (Sigma-Aldrich), and plates were read at 405 nm.
To analyze T cell–dependent humoral responses, mice received two IP injections with 10 µg hen egg OVA (Sigma-Aldrich) emulsified in 50% CFA (day 0) and IFA (day 14). For T cell–independent responses, mice received two IP injections (days 0 and 14) with 50 µg of 2,4, Dinitrophenyl-Amino-Ethyl-Carboxy-Methyl-Ficoll (DNP-Ficoll; Biosearch Technologies) emulsified in 50% IFA. Blood samples were collected (day 7 and 24) and antigen-specific antibody titers were determined in polycarbonate 96 multiwell plates (Maxisorp; Nunc) coated overnight at 4°C with OVA or albumin-DNP (Sigma-Aldrich) solution (10 µg/ml), in carbonate buffer.
To analyze T cell–dependent humoral responses, mice received two IP injections with 10 µg hen egg OVA (Sigma-Aldrich) emulsified in 50% CFA (day 0) and IFA (day 14). For T cell–independent responses, mice received two IP injections (days 0 and 14) with 50 µg of 2,4, Dinitrophenyl-Amino-Ethyl-Carboxy-Methyl-Ficoll (DNP-Ficoll; Biosearch Technologies) emulsified in 50% IFA. Blood samples were collected (day 7 and 24) and antigen-specific antibody titers were determined in polycarbonate 96 multiwell plates (Maxisorp; Nunc) coated overnight at 4°C with OVA or albumin-DNP (Sigma-Aldrich) solution (10 µg/ml), in carbonate buffer.
8
Quantification of HSV-2 gB Antibodies
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Prokaryotic expression of HSV-2 gB protein was diluted with PBS to a concentration of 2.0 μg/mL and used for 96-well plate coating at 50 μL per well overnight at 4 °C. Briefly, the double-diluted samples collected on day 14 post boost were added to the plates and measured (the starting dilution ratio for sera was 1/5000, and that for vaginal fluids was 1/200) with endpoint ELISA assay. The values of the color intensity were read by an automated microplate ELISA reader (Tecan) at the wavelengths of A450 and A630 (for reference). The endpoint titers of gB-specific Igs were calculated using a combination of Excel and GraphPad Prism 8.0.2 software [14 (link)]. In the same dilution, murine samples read by OD450 were considered positive if they were higher than the OD450 of the negative control.
The concentration values of gB-specific isotype Igs in murine sera were measured by HRP-conjugated anti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgM (the starting dilution ratio for sera was 1/5000; Southern Biotechnology Associates, Birmingham, AL, USA) according to the manufacturer’s instructions. The Ig isotype concentration was calculated according to the respective standard curve.
The concentration values of gB-specific isotype Igs in murine sera were measured by HRP-conjugated anti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgM (the starting dilution ratio for sera was 1/5000; Southern Biotechnology Associates, Birmingham, AL, USA) according to the manufacturer’s instructions. The Ig isotype concentration was calculated according to the respective standard curve.
9
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.
10
ELISPOT Assay for Antigen-Specific Antibodies
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Single cell suspension was obtained from spleen, lymph nodes, bone marrow, synovium and resuspended in complete RPMI (ThermoFisher, #61870044) media containing 10% FCS (ThermoFisher, #26140079) and penicillin/streptomycin (Sigma, #P4333). Then, cells were added into COL2-coated (10 μg/ml), dsDNA-coated (20 μg/ml, Sigma-Aldrich, #D3664), nucleosome-coated (10 μg/ml, homemade) or anti-IgG-coated (1 μg/ml) ELISPOT plates (Merck Millipore, #MSIPS4W10). For dsDNA coating, Poly-l -Lysine (20 μg/ml, #P2658) was pre-coated one day before and dsDNA was coated in sterile TE buffer. After 2 h incubation at 37 °C, plates were washed and detected by biotinylated goat anti-mouse IgG (Southern Biotech, #1030-08), IgG1(Southern Biotech, #1070-08) or IgG2b (Southern Biotech, #1090-08), followed by ExtrAvidin® conjugated alkaline phosphatase (Sigma-Aldrich, #E2636) and BCIP/NBT (Sigma-Aldrich, #B5655). Spots were scanned with ImmunoScan and analyzed with ImmunoSpot software (Cellular Technology Ltd.).
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