Anti igg3

Manufactured by Southern Biotech

Sourced in United States

Anti-IgG3 is a laboratory reagent used to detect and quantify the presence of IgG3 antibodies in biological samples. It functions by binding to and labeling IgG3 molecules, allowing for their identification and measurement.

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Lab products found in correlation

Anti igg2b, by Southern Biotech (3 mentions) Anti igg4, by Southern Biotech (2 mentions) Lsm 510 meta confocal microscope, by Zeiss (2 mentions) 3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions) Biotinylated secondary antibody, by Southern Biotech (1 mentions) Liquichip ni nta beads, by Qiagen (1 mentions) Anti igg2c, by Southern Biotech (1 mentions) 3 3 5 5 tetramethylbenzidine substrate, by Thermo Fisher Scientific (1 mentions) Super aquablue elisa substrate, by Thermo Fisher Scientific (1 mentions) Np bsa, by Biosearch Technologies (1 mentions) β galactosidase, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Biosearch Technologies (1 mentions) Biotinylated goat anti mouse iga, by BD (1 mentions) Asys expert 96 elisa reader, by Harvard Bioscience (1 mentions) Elisa plate, by Thermo Fisher Scientific (1 mentions) Anti b220, by Thermo Fisher Scientific (1 mentions) Cy3 labeled goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions) Apc labeled anti b220, by Thermo Fisher Scientific (1 mentions) Non fat milk, by Merck Group (1 mentions) Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)

8 protocols using anti igg3

Measurement of Mf-specific Antibodies

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Mf-specific immunoglobulin levels were measured by ELISA [5] (link). Briefly 96-well plates were coated overnight at 4°C with 1 µg/ml soluble Mf extract (MfAg) in 50 µl carbonate buffer (pH 9.6). After blocking each well with 10% FCS in carbonate buffer, the plates were incubated with individual mouse sera diluted 1∶50 in PBS 0.5% Tween-20. Antigen-specific antibodies were detected using horseradish peroxidase (HRP)-conjugated goat anti-IgM (Southern Biotechnology Associates, Birmingham, AL, USA; SBA 1020-05), anti-IgG1 (SBA1070-05), anti-IgG2a (SBA 1080-05), anti-IgG2b (SBA 1090-05) or anti-IgG3 (SBA 1100-05). 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma) was used as the substrate. Plates were read at 405 nm. Total IgE was measured by ELISA as previously described [44] (link).

Cadman E.T., Thysse K.A., Bearder S., Cheung A.Y., Johnston A.C., Lee J.J, & Lawrence R.A. (2014). Eosinophils Are Important for Protection, Immunoregulation and Pathology during Infection with Nematode Microfilariae. PLoS Pathogens, 10(3), e1003988.

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Quantifying Anti-DSG3 Antibody IgG Subclasses

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Sera from patients were analyzed before treatment at baseline, after treatment, and at the time of relapse, if applicable. To detect and quantify anti-DSG3 Ab IgG subclasses, we developed an ALBIA-DSG3, which consisted of coupling human recombinant DSG3 protein to fluorescent beads (LiquiChip Ni-NTA Beads; Qiagen, Hilden, Germany) according to the manufacturer’s protocol. To determine the isotype of serum anti-DSG3 Abs, DSG3-coated beads were incubated with sera diluted at 1:150, then incubated with anti-IgG1 (1:125), anti-IgG2 (1:125), anti-IgG3 (1:200), or anti-IgG4 (1:200) biotinylated secondary antibody (SouthernBiotech, Birmingham, AL, USA), and finally with streptavidin–R-phycoerythrin (Qiagen). The mean fluorescence intensity (MFI) was determined on a Bio-Plex apparatus using Manager software version 4.0 (Bio-Rad, Hercules, CA, USA). Negative control with no serum and positive control [anti-DSG3 Calibrator of ELISA kit (EUROIMMUN)] were included in every assay. The anti-DSG3 Ab serum levels were determined with the formula (MFI^serum/MFI^Calibrator) × 100, in which the calibrator was the anti-DSG3-positive control previously mentioned that was used on every 96-well plate and set arbitrarily to 100 arbitrary units (AU). For each isotype, we considered a positivity threshold corresponding to + 3 standard deviations relative to the mean value obtained from the sera of 36 HD.

Golinski M.L., Lemieux A., Maho-Vaillant M., Barray M., Drouot L., Schapman D., Petit M., Hertl M., Boyer O., Calbo S., Joly P, & Hébert V. (2022). The Diversity of Serum Anti-DSG3 IgG Subclasses Has a Major Impact on Pemphigus Activity and Is Predictive of Relapses After Treatment With Rituximab. Frontiers in Immunology, 13, 849790.

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Serum Antibody Profiling by ELISA

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Serum was collected by submandibular bleed and microcentrifugation at >18,000 g for 30 min at 4°C. Half area high-binding 96-well plates (Costar) were used for all assays. For total Ig at steady state, plates were coated with goat anti-Ig (2 µg/ml; SouthernBiotech) overnight in coating buffer bicarbonate/carbonate coating buffer (100 mM, pH 9.6). For NP-specific antibody ELISAs, plates were coated with NP-BSA (5 μg/ml; LGC Biosearch Technologies). Diluted serum was added overnight at 4°C or at room temperature for 2 h and detected with biotinylated goat anti-IgG1, anti-IgG3, anti-IgG2b, anti-IgG2c, anti-IgA, or anti-IgM (2 µg/ml; SouthernBiotech) followed by HRP-avidin (1:5,000; SouthernBiotech) and developed with Super AquaBlue ELISA substrate or 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Fisher Scientific) with the manufacturer’s recommended absorbance. For total IgE, the process was the same, except that plates were coated with anti-IgE (2 µg/ml, clone R35-72) and detected with biotinylated anti-IgE (2 µg/ml, clone R35-118).

Wigton E.J., Mikami Y., McMonigle R.J., Castellanos C.A., Wade-Vallance A.K., Zhou S.K., Kageyama R., Litterman A., Roy S., Kitamura D., Dykhuizen E.C., Allen C.D., Hu H., O’Shea J.J, & Ansel K.M. (2021). MicroRNA-directed pathway discovery elucidates an miR-221/222–mediated regulatory circuit in class switch recombination. The Journal of Experimental Medicine, 218(11), e20201422.

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ELISA for Mouse Antibody Isotypes

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ELISA plates (Nunc) were coated with 2 µg/ml β-Galactosidase (Sigma) or 500 ng/well of NP(30) conjugated with BSA (Biosearch Technologies). To detect IgM, IgG, IgG1, IgG2b, IgG2c, IgG3 or IgA plates were coated with unconjugated goat anti-mouse IgM, IgG or IgA (Southern Biotech). After incubation overnight (4°C), washing with PBS + 0.05% Tween20 and blocking for 1 h with PBS containing 2% dry milk (blocking buffer), 5 μl of serum was added in a total volume of 150 μl, followed by 3-fold serial dilutions in blocking buffer and incubated for 2 h at room temperature (RT). Plates were washed six times, and primary antibodies, biotinylated goat anti-mouse IgM, or goat anti-mouse IgG (both from Mabtech AB), biotinylated goat anti-mouse IgA (BD Pharmingen), or HRP-coupled anti-IgG1, anti-IgG2b, anti-IgGc or anti-IgG3 (Southern Biotech) were added in 100 μl PBS/well followed by incubation for 1.5 h, at RT. Streptavidin-HRP was added to biotinylated antibodies in 100 μl PBS/well after washing six times and incubated for 1 h, at RT. The assay was developed with TMB substrate (KPL), the reaction was stopped with 1 M H₂SO₄, and the OD was read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom Ltd.).

Khoenkhoen S., Ádori M., Solís-Sayago D., Soulier J., Russell J., Beutler B., Pedersen G.K, & Karlsson Hedestam G.B. (2022). IκBNS expression in B cells is dispensable for IgG responses to T cell-dependent antigens. Frontiers in Immunology, 13, 1000755.

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Immunofluorescence of IDO1 in Splenic Sections

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Spleens were harvested from the mice after NP-Ficoll immunization at indicated times, snap-frozen in liquid nitrogen and frozen in Tissue-Tek OCT compound (Sakura) at −80°C. For immuno-fluorescence of IDO1 5μm frozen splenic sections were immediately fixed in −20°C methanol. The sections were blocked with PBS containing 1% nonfat milk (Sigma) and stained with 4μg/mL polyclonal rabbit anti-mouse IDO1 (a gift from Dr. David Munn) in PBS containing 1% nonfat milk. After extensive washing with TBS + 0.05% tween 20 (ACROS Organics), the sections were stained with a 1:400 dilution of CY3-labeled goat anti-rabbit IgG (Jackson Immunoresearch), APC-labeled anti-B220 (Ebioscience), or FITC-labeled anti-CD138 (BD Pharmingen). To examine antibody response to NP-Ficoll immunization, sections were incubated with anti-IgM (BioLegend) and anti-IgG3 (Southern Biotech) in blocking buffer for 60 minutes at room temperature in the dark. For stimulated and unstimulated purified B cells, cytospins were prepared and fixed with −20°C methanol. Cells were made permeable using Perm Buffer III (BD Biosciences) and stained for IDO1 as explained above and anti-B220 (eBioscience). Sections and cytospins were mounted with Prolong Gold anti-fade with DAPI (Invitrogen). Fluorescent images were captured using a Zeiss LSM 510 Meta confocal microscope equipped with 405-, 488-, 561-, and 633-nm lasers.

Shinde R., Shimoda M., Chaudhary K., Liu H., Mohamed E., Bradley J., Kandala S., Li X., Liu K, & McGaha T.L. (2015). B cell-intrinsic Indoleamine 2,3-dioxygenase1 regulates humoral immunity to T cell independent antigens. Journal of immunology (Baltimore, Md. : 1950), 195(5), 2374-2382.

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ELISA for Mouse Immunoglobulin Isotypes

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Maxisorp plates were coated using one of the following Abs: anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgG3, anti-IgM (Southern Biotech). After incubation with mouse sera, revelation was performed using biotin-coupled anti-mouse immunoglobulin (Ig) (H + L) (Jackson IR), streptavidin HRP (ThermoFisher), TMB, and H₂SO₄. Optical densities were assessed using a ThermoScientific Multiskan FC apparatus.

Briet C., Bourdenet G., Rogner U.C., Becourt C., Tardivel I., Drouot L., Arnoult C., do Rego J.C., Prevot N., Massaad C., Boyer O, & Boitard C. (2017). The Spontaneous Autoimmune Neuromyopathy in ICOSL−/− NOD Mice Is CD4+ T-Cell and Interferon-γ Dependent. Frontiers in Immunology, 8, 287.

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ELISPOT Assay for IgM ASCs

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IgM ASCs were enumerated as described previously (Doucett et al., 2005 (link)). In brief, anti-IgM (331) or anti-IgG3 (SouthernBiotech) was coated overnight onto 96-well plates (Multi-Screen HA Filtration, EMD Millipore). Plates were blocked with PBS/4% BSA. Live cells counted using Trypan blue exclusion were either serially diluted twofold into the ELISPOT plate in culture media (RPMI 1640 [Gibco], 10% heat-inactivated fetal bovine serum, 292 µg/ml l-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-mercaptoethanol) or were directly deposited by FACS into culture media–containing wells. Plates were incubated overnight at 37°C/5% CO₂. IgM binding was identified with biotinylated anti-IgM (SouthernBiotech), biotinylated anti-IgM^a (BD), biotinylated anti-IgM^b (BD), or biotinylated anti-IgG3 (SouthernBiotech), followed by Streptavidin–Horseradish Peroxidase (Vector Laboratories) and the substrate 3-amino-9-ethylcarbazole (Sigma-Aldrich). Spots were enumerated, and spot sizes, expressed as the number of pixels, were determined using the AID EliSpot Reader System (Autoimmun Diagnostika). Total BM spots were calculated based on the assumption that one femur = 12.7% of total BM cells (Benner et al., 1981 (link)).

Savage H.P., Yenson V.M., Sawhney S.S., Mousseau B.J., Lund F.E, & Baumgarth N. (2017). Blimp-1–dependent and –independent natural antibody production by B-1 and B-1–derived plasma cells. The Journal of Experimental Medicine, 214(9), 2777-2794.

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BG505 gp120 antibody detection

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In brief, 50 μg of the recombinant BG505 clade A gp120 (donated by Elise Landais) were used to coat plates prior to incubation with diluted test plasma. Subclass-specific antihuman immunoglobulin conjugated to alkaline phosphatase (mouse anti-IgG, anti-IgG₁, anti-IgG₂, anti-IgG₃, anti-IgG₄ or goat anti-IgA) from Southern Biotech, USA were used for antibody subclass detection. Relative arbitrary antibody units were calculated by interpolating OD readings to standard curves prepared using pooled hyper-reactive HIV-positive plasma.

Nduati E.W., Gorman M.J., Sein Y., Hermanus T., Yuan D., Oyaro I., Muema D.M., Ndung’u T., Alter G, & Moore P.L. (2021). Coordinated Fc-effector and neutralization functions in HIV-infected children define a window of opportunity for HIV vaccination. AIDS (London, England), 35(12), 1895-1905.

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