Shrimp-, lobster-, crab-, walnut-, and pecan-specific IgE, IgG, IgG1, IgG2a were quantified by ELISA, as previously reported (69 (link)). Briefly, plates were coated with 20 µg/ml food extracts (for samples) or 20 µg/ml HSA-DNP (for standard curves). After blocking with 2% BSA in PBS-0.05% Tween, serum samples were diluted 1:100 for IgE, 1:5,000 for IgG, 1:20,000 for IgG1 and 1:1,000 for IgG2a. Standard curves of mouse IgE anti-DNP, IgG1 anti-DNP or IgG2a anti-DNP (Accurate Chemicals, Westbury, NY) were generated ranging from 0.002–2 µg/ml. For IgE plates, the following detection antibodies were used in succession: 0.5 µg/ml sheep IgG anti-mouse IgE (The Binding Site, Birmingham, UK), 0.5 µg/ml biotinylated donkey IgG anti-sheep IgG (Accurate Chemicals), and 0.5 µg/ml NeutrAvidin-HRP (Pierce Biotechnology, Rockford, IL). For IgG, IgG1, and IgG2a plates, HRP goat anti-mouse IgG (Invitrogen, Waltham, MA), anti-mouse IgG1-HRP (Southern Biotech, Birmingham, AL), or anti-mouse IgG2a-HRP (Southern Biotech) were used, respectively. All plates were developed with TMB (SeraCare, Milford, MA), stopped with 1% HCl (SeraCare), and read on a plate spectrophotometer (BioTek, Winooski, VT) at 450 nm. Antigen-specific IgE, IgG1, and IgG2a concentrations were calculated based on the standard curve and dilution factor. Antigen-specific IgG is presented as O.D. values.
Anti mouse igg1 hrp
Manufactured by Southern Biotech
Sourced in United States
Anti-mouse IgG1-HRP is a laboratory reagent used as a secondary antibody. It is conjugated with horseradish peroxidase (HRP), an enzyme that can be used for detection in various immunoassay techniques.
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Lab products found in correlation
Anti mouse iga hrp, by Southern Biotech (5 mentions)
Anti mouse igg2c hrp, by Southern Biotech (5 mentions)
Anti mouse ige hrp, by Southern Biotech (4 mentions)
Anti mouse igg2a hrp, by Southern Biotech (3 mentions)
Igg2a hrp, by Southern Biotech (3 mentions)
Anti mouse igg hrp, by Southern Biotech (3 mentions)
Igg2b hrp, by Southern Biotech (2 mentions)
Elx808, by Agilent Technologies (2 mentions)
Elisa plate, by Thermo Fisher Scientific (2 mentions)
Anti syk, by Cell Signaling Technology (2 mentions)
Anti rabbit igg hrp, by GE Healthcare (2 mentions)
Anti phosphorylated syk, by Cell Signaling Technology (2 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (2 mentions)
Anti goat igg hrp, by Santa Cruz Biotechnology (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti p erk1 2, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Plate spectrophotometer, by Agilent Technologies (1 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by LGC (1 mentions)
19 protocols using anti mouse igg1 hrp
1
Quantification of Food-Specific Antibodies
2
Monoclonal Antibody Purification and Isotyping
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Example 2
The monoclonal hybridoma cells (chA1 clone) were subcultured in vitro and the released mAb (chA1-L1) was purified from culture supernatant with the Pierce Thiophilic Adsorption Kit (Thermo Scientific).
Isotype discrimination was performed by ELISA assay to determine the class and subclass of the chA1-L1 monoclonal antibody. The following anti-mouse secondary antibodies were used: anti-mouse IgM-HRP (1:20000; Dianova); anti-mouse IgG1-HRP, IgG2a-HRP, IgG2b-HRP and IgG3-HRP (1:6000; Southern Biotech); anti-mouse IgG (H+L)-HRP (1:20000; Abcam). Results showed that the antibody is an IgG2a isotype (data not shown).
3
HDM-specific Humoral and Cellular Responses
4
SARS-CoV-2 Antibody Quantification in Mice and Humans
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A semi-quantitative ELISA was used to determine the levels of IgG, IgG1, IgG2a, and IgA antibodies in the sera of vaccinated mice. Also, the levels of IgG and IgA antibodies were determined in the sera of recovered COVID-19 patients.
First, high binding ELISA plates (Biomat, Italy) were coated with SARS-CoV-2 S- protein recombinant antigen (Sigma-Aldrich) at 1 μgmL−1. After washing plates, 50 μL of diluted serum samples collected from immunized mice and recovered COVID-19 patients were separately added to wells. After incubation for 1 h at 37 °C, plates were washed with PBS and then 100 μL of the following secondary antibody was separately added: 1) anti-mouse IgG-HRP, 2) anti-mouse IgG1-HRP, 3) anti-mouse IgG2a-HRP, 4) anti-mouse IgA-HRP, 5) anti-human IgG-HRP, 6) anti-human IgA-HRP (Southern Biotech). After incubation and washing with PBS, 50 μL of 3,3′, 5,5′-tetramethylbenzidine was added and then the reactions was stopped by adding 50 μL of 10% sulfuric acid. Finally, the absorbance of each well was read by a Spectrophotometer at 450 nm (BioTek Industries) and serum levels of antibodies was measured using a standard curve.
First, high binding ELISA plates (Biomat, Italy) were coated with SARS-CoV-2 S- protein recombinant antigen (Sigma-Aldrich) at 1 μgmL−1. After washing plates, 50 μL of diluted serum samples collected from immunized mice and recovered COVID-19 patients were separately added to wells. After incubation for 1 h at 37 °C, plates were washed with PBS and then 100 μL of the following secondary antibody was separately added: 1) anti-mouse IgG-HRP, 2) anti-mouse IgG1-HRP, 3) anti-mouse IgG2a-HRP, 4) anti-mouse IgA-HRP, 5) anti-human IgG-HRP, 6) anti-human IgA-HRP (Southern Biotech). After incubation and washing with PBS, 50 μL of 3,3′, 5,5′-tetramethylbenzidine was added and then the reactions was stopped by adding 50 μL of 10% sulfuric acid. Finally, the absorbance of each well was read by a Spectrophotometer at 450 nm (BioTek Industries) and serum levels of antibodies was measured using a standard curve.
5
HDM-specific Immunoglobulin Responses
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HDM-specific IgG1 and IgE were measured by enzyme-linked immunosorbent assay (ELISA) in the serum of HDM-treated mice. Secondary Abs used were anti-mouse IgG1-HRP (Southern Biotech, cat. no.1070–05) and anti-mouse IgE-HRP (Southern Biotech, cat. no.1110–05). To measure HDM-specific T-cell responses, lung cells were restimulated with 25 µg/ml of HDM for 4 days. IL-5 cytokines were measured in the supernatant by ELISA.
6
HDM-Specific Antibody and T Cell Responses
7
Measurement of NP-specific IgG1 by ELISA
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NP-specific IgG1 was measured by ELISA. In brief, NP25-BSA (Biosearch Technologies) was coated on ELISA plates (Immulon 4HBX; Thermo Fisher) overnight. The plates were then washed with ELISA wash buffer (0.5% Tween in PBS), serially diluted sera were applied to the plates, and plates were incubated 2 h at room temperature. Anti–mouse IgG1 HRP (Southern Biotech) was used to detect NP-specific IgG1. To measure IgG1 affinity maturation, antibody titer was determined with NP4-BSA and NP25-BSA. For allotype-specific IgG1 detection, anti–mouse IgG1a or IgG1b-biotin antibody (BD Biosciences) was used and followed by a streptavidin-HRP (BD Biosciences) reaction. After a wash step, 2,2'-Azino-di-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) substrate (KPL) was added to the wells, and enzyme reaction was stopped by ABTS HRP Stop Solution (KPL). Optical density at 405 nm was measured with Fluostar Optima plate reader and software (BMG Labtech).
8
Antigen-specific antibody response analysis
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Blood was collected from the retro-orbital sinus two weeks after the second immunization and sera prepared. Sera were stored at 4°C until antigen-specific antibody responses were analyzed by ELISA. Briefly, ELISA plates (Nunc, Rochester, NY) were coated with 1 μg/ml antigen in 0.1 M bicarbonate buffer and blocked with 0.1% BSA-PBS. Following washes in PBS/Tween, serially diluted serum samples were added. After incubation and further washes, either anti-mouse IgG-HRP, anti-mouse-IgG2c-HRP or anti-mouse IgG1-HRP were added (all Southern Biotech, Birmingham, AL). After incubation and washing, ABTS-H2O2 (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added to the plates to reveal any reactions, which were stopped by the addition of 0.1 N H2SO4. Plates were analyzed at 405 nm (ELX808, Bio-Tek Instruments Inc, Winooski, VT). Midpoint titers were determined as EC50 values from weighted curve fits using the GraphPad Prism package V 6.03.
9
Serum IgG Subclass Evaluation
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The serum levels of IgG2a and IgG1 were evaluated using HRP-conjugated antibodies against IgG2a and IgG1. To this aim, Maxi-Sorp plates (Greiner, Germany) were coated with 10 μg/ml L. major F/T Ag overnight and then blocked with 1% BSA at room temperature for 2 hours. After that, 100 μl (at a dilution of 1:50) of serum was added and incubated at 37°C for about 2 hours. The plates were washed, and anti-mouse IgG1-HRP or IgG2a-HRP (1: 10,000; Southern-Biotech, Canada) was added to each plate. After 2 h incubation at 37°C, the plates were rinsed again, and 20 μl of the TMB micro-well peroxidase substrate solution (KPL, Gaithersburg, MD) was added to each sample. Finally, the plates were incubated for 20 minutes at 37°C, and the reaction was blocked by adding 1% sodium dodecyl sulfate. The absorbance of each group was measured at a wavelength of 405 nm.
10
SARS-CoV-2 Spike Protein Antibody Assay
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Serum samples were collected on the indicated days and stored in BD Vacutainers; 96-well plates were coated with 2 μg/mL of either recombinant SpikeRBD or SpikeRBD (K417T, E484K, N501Y) proteins in phosphate-buffered saline (PBS) overnight at 4 °C and incubated in blocking buffer (0.05% Tween20 + 2% bovine serum albumin in PBS). Plates were incubated with diluted serum samples for 2 h at room temperature. Plates were then washed four times with PBS and incubated with goat anti-mouse IgG-HRP, anti-mouse IgM-HRP, anti-mouse IgG1-HRP, or IgG2b-HRP (SouthernBiotech) at 1:10,000 or with IgA-HRP at 1:2,000 in blocking buffer for 1 h. Plates were developed with 3,3',5,5'-tetramethylbenzidine liquid substrate reagent (Sigma). The reaction was stopped with 1 N HCl, and absorbance was read at 450 nm.
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