The DENV binding antibody titer was analyzed by indirect ELISA. 96-well plates (Thermo Scientific) were coated with recombinant protein in a coating buffer (0.1 M NaHCO3) and incubated overnight at 4°C. The following day, plates were blocked with 3% BSA in PBS for 2 h at RT. Triplicate samples of serially diluted plasma ranging from 1:100 to 1:500,000 were added to the plate and incubated for 2 h at RT or overnight at 4°C. After washing, secondary anti-IgG (Sigma), anti-IgG1(Invitrogen), and anti-IgG2a (Invitrogen) or IgG2c (Abcam) antibodies conjugated with HRP were added at 1:2,000 (IgG, IgG1, IgG2a) and 1:5,000 (IgG2c) dilution for 1 h at RT. The plates were then developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Sigma) for 15–20 min. The reaction was stopped with a stop buffer (Invitrogen), and the optical density (OD) measured at 450 nm. Cut-off values for each dilution were set using the OD of naive samples in the formula: naive OD at a dilution + (2.5 × standard deviation). Starting from the lowest dilution, the sample dilution prior to the one that was exceeded by the cut-off was considered to be the end titer value.89
Igg2c
Manufactured by Abcam
Sourced in United States
IgG2c is an immunoglobulin G (IgG) subclass that is found in the blood and plays a role in the immune system. It functions as an antibody, binding to and neutralizing foreign substances or pathogens.
Automatically generated - may contain errors
Lab products found in correlation
Igg2b, by Abcam (1 mentions)
Anti human fviii igg2a, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Elisa reader, by Tecan (1 mentions)
96 well plate, by Corning (1 mentions)
Horseradish peroxidase conjugatedgoat anti mouse igg h l, by Abcam (1 mentions)
Igg2a, by BD (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Igg2a, by Abcam (1 mentions)
Alkaline phosphatase substrate, by Merck Group (1 mentions)
Alkaline phosphatase conjugated anti mouse ab specific for igg, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Sodium carbonate, by Merck Group (1 mentions)
Igg2b r12 3, by BD (1 mentions)
A85 1, by BD (1 mentions)
Avidin hrp, by BD (1 mentions)
7 protocols using igg2c
1
Determining DENV Antibody Titers by ELISA
2
Anti-FVIII IgG Subclass Quantification
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IgG subclass of anti-FVIII antibodies was assessed using an in-house established ELISA assay. Briefly, 96-well plates (Corning) were coated overnight with 1 µg/mL rhFVIII in PBS at room temperature. Plates were blocked using 1% bovine serum albumin (Sigma-Aldrich) in PBS. Standards or plasma samples were diluted in 1% bovine serum albumin in PBS. Anti-FVIII IgG isotype-specific antibodies were detected by anti-mouse horseradish peroxidase–conjugated IgG1, IgG2a (both from Southern Biotech, Birmingham, AL), IgG2b, or IgG2c (both from Abcam) and 3,3',5,5'-tetramethylbenzidine substrate solution (Perbio Science). Absorbance was detected at 450 nm using an ELISA reader (Tecan). The concentration of anti-FVIII isotype–specific antibodies was estimated from a standard curve obtained using serial dilutions of mouse anti-human FVIII IgG1 (Merck Millipore), anti-human FVIII IgG2a (Thermo Fisher Scientific) starting at 50 ng/mL, or mouse anti-FVIII reference plasma starting at 50 arbitrary units per milliliter.
3
Quantifying Anti-Muc1 Antibody Responses
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ELISA
was used for the analysis of induced anti-Muc1 antibodies in antiserum.
96-well MaxiSorp plates were coated by incubating overnight at 4 °C
with 1 μg/mL glycopeptide 11 in BSA, washed three times with
washing buffer (PBS containing 0.05% Tween-20), and blocked for 30
min at 37 °C with blocking buffer (washing buffer supplemented
with 1% BSA). Dilutions of sera in blocking buffer were added and
incubated for 2 h at 37 °C. The plates were washed three times
with washing buffer and incubated with the different antibody subtypes
IgG1, IgG2b (BD Pharmigen), IgG2a (PharMingen), IgG2c (Abcam), and
IgG3 (BioLegend). After incubation and three washes, horseradish peroxidase-conjugated
goat anti-mouse IgG (H + L) (Abcam) diluted 1:1000 in blocking buffer
was added for 1 h at 37 °C. The plates were then washed three
times and incubated for 10 min with 50 μL per well of 2,2′-azino-di-(3-ethylbenzothiazoline
sulfonic acid substrate in citrate buffer (pH 4.4 and 1:4000) 30%
H2O2. The reaction was quenched by adding 50
μL of 1 M aqueous sulfuric acid to each well. Plates were read
at 410 nm using a Tecan Infinite F200 Pro microplate reader. For each
IgG of the antiserum, optical densities were measured and the absorbance
curves were fitted to obtain antibody titers. There were no IgGs detectable
for the negative controls (untreated mice).
was used for the analysis of induced anti-Muc1 antibodies in antiserum.
96-well MaxiSorp plates were coated by incubating overnight at 4 °C
with 1 μg/mL glycopeptide 11 in BSA, washed three times with
washing buffer (PBS containing 0.05% Tween-20), and blocked for 30
min at 37 °C with blocking buffer (washing buffer supplemented
with 1% BSA). Dilutions of sera in blocking buffer were added and
incubated for 2 h at 37 °C. The plates were washed three times
with washing buffer and incubated with the different antibody subtypes
IgG1, IgG2b (BD Pharmigen), IgG2a (PharMingen), IgG2c (Abcam), and
IgG3 (BioLegend). After incubation and three washes, horseradish peroxidase-conjugated
goat anti-mouse IgG (H + L) (Abcam) diluted 1:1000 in blocking buffer
was added for 1 h at 37 °C. The plates were then washed three
times and incubated for 10 min with 50 μL per well of 2,2′-azino-di-(3-ethylbenzothiazoline
sulfonic acid substrate in citrate buffer (pH 4.4 and 1:4000) 30%
H2O2. The reaction was quenched by adding 50
μL of 1 M aqueous sulfuric acid to each well. Plates were read
at 410 nm using a Tecan Infinite F200 Pro microplate reader. For each
IgG of the antiserum, optical densities were measured and the absorbance
curves were fitted to obtain antibody titers. There were no IgGs detectable
for the negative controls (untreated mice).
4
Evaluating CMFO-DMT Immunological Mechanisms
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To evaluate immunological mechanisms of CMFO-DMT, the CMFO-specific antibodies IgG, IgG1, and IgG2c (Cat#151276, 133045, 157720; Abcam, Cambridge, MA, USA) in the sera of mice vaccinated with PBS, DMT, BCG, or CMFO-DMT were determined by ELISA at week 10 after immunization. The results are expressed as the mean ± standard error of the mean (SEM) log10 endpoint titers per group (n = 6), as previously described (Wang et al., 2015 (link)).
5
ELISA-based Anti-Ft Antibody Quantification
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Anti-Ft Ab production in response to immunization and/or Ft infection in immunized mice was measured through enzyme linked immunosorbent assay (ELISA). Briefly, ELISA plates were coated overnight at 4°C with 50 μl/well of inactivated Ft SchuS4 [5x107 CFU/ml in carbonate buffer [4.3g/l sodium bicarbonate and 5.3 g/l sodium carbonate (Sigma-Aldrich, St. Louis, Missouri) at pH 9.4]. Plates were then blocked at 4°C for 2 hours with 200 μl/well of PBS containing 5% BSA and 0.02% sodium azide. Subsequently three-fold dilutions of sera (starting with 1:50) were added to the plates (50 μl/well) and incubated for 2 hours at 4°C. After washing, alkaline phosphatase conjugated anti-mouse Ab specific for IgG, IgA (Sigma-Aldrich, St. Louis, Missouri), or IgG2c (Abcam, Cambridge, MA), were added and incubated for 1 hours at 4°C. Following secondary Ab incubation and washing alkaline phosphatase substrate (Sigma-Aldrich, St. Louis, Missouri) of 100 μl/well was added and plates were read at 405 nm using microplate reader following a 5-second (sec) shake.
6
Quantifying Serum Antibody Levels
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Antibody titers of SRBC-specific Ig and total Ig in serum were measured by ELISA, similar to previously reported [26 (link)]. For anti-SRBC Ab analysis, 96 well Nunc-Immuno plates were coated with SRBC membrane protein overnight at 4°C. For anti-mouse IgA, anti-mouse total IgG or anti-mouse IgM Abs, directly peroxidase-conjugated secondary Abs (Sigma-Aldrich) were used. For analysis of IgG1 (A85-1, BD Biosciences), IgG2b (R12-3, BD Biosciences), IgG2c (Abcam) and IgG3 (R40-82, BD Biosciences), biotin-labeled secondary Abs were used along with Avidin-HRP (BD Biosciences). For total Ig isotype analysis, 96 well Nunc-Immuno plates were coated with anti-Ig kappa light chain Ab (187.1, BD Biosciences) diluted in PBS overnight at 4°C. Wells were blocked with 10% FCS and diluted serum was added and incubated at room temperature for 2 h.
7
ELISA for Anti-Collagen II Antibodies
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100 μl of 10 μg/ml CII in 0.1 M Na2CO3 was added to the wells of a 96-well plate and incubated at 4 °C overnight. The wells were washed 3 times with 300 μl of PBS containing 0.05% polysorbate 20 and incubated with 300 μl of PBS containing 1% bovine serum albumin (BSA) for 1 h. After washing the wells, 100 μl of sera diluted 5000 times with PBS containing 1% BSA was added to each well and incubated for 1 h at room temperature. After washing the wells, 100 μl of HRP-conjugated anti-mouse IgG, IgG1(Bethyl laboratories, TX, USA), or IgG2c (Abcam, Cambridge, UK) antibody diluted 5000 times with 1% BSA in PBS was added and incubated for 1 h at room temperature. After washing the wells, 100 μl of 3,3’,5,5’-tetramethylbenzidine was added to each well. After incubation for 5 to 10 min, 50 μl of 1 M H2SO4 was added, and the optical density was read at 450 nm using a microplate reader.
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