Antibody titers were determined using an ELISA. Two μg/ml of recombinant Ebola virus GP was plated in a flat bottom 96 well plate overnight. Plates were incubated with blocking buffer (5% milk, 0.05% Tween in PBS) for 2 h, and then serum samples were added to plates. The standard protocol used half log dilutions starting at a 1:100 dilution. After 2 h, plates were washed with PBS + 0.05% Tween and secondary antibody was added at a 0.6 μg/ml. Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05). One hour later, plates were washed and exposed using Sure Blue TMB 1-component substrate and stop solution (KPL), and the absorbance at 450 nm was recorded. Serum from unvaccinated animals was used to establish background and titers were defined as the serum dilution resulting in an absorbance greater than 0.2, where background was universally less than 0.2. Serum from animals previously determined to contain anti-GP antibody was included in each assay to serve as a positive control.
Igg1 hrp
Manufactured by Southern Biotech
Sourced in United States
IgG1-HRP is a laboratory reagent that consists of horseradish peroxidase (HRP) conjugated to immunoglobulin G1 (IgG1) antibodies. IgG1-HRP can be used as a detection or labeling agent in various immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Igg2c hrp, by Southern Biotech (8 mentions)
Igg3 hrp, by Southern Biotech (7 mentions)
Goat anti mouse igg hrp, by Southern Biotech (4 mentions)
Igg2b hrp, by Southern Biotech (3 mentions)
Igg2a hrp, by Southern Biotech (3 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Goat anti mouse ige unlb, by Southern Biotech (2 mentions)
Rabbit anti goat igg ap, by Thermo Fisher Scientific (2 mentions)
Elisa for il 13, by Thermo Fisher Scientific (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Synergy ht microplate reader, by Agilent Technologies (2 mentions)
96 well flat bottom plate, by Thermo Fisher Scientific (2 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (2 mentions)
Monoclonal anti mouse iga hrp, by Southern Biotech (2 mentions)
Hrp conjugated ig subclass antibody, by Southern Biotech (2 mentions)
Igg2c, by Southern Biotech (2 mentions)
Goat anti mouse igm hrp, by Southern Biotech (2 mentions)
18 protocols using igg1 hrp
1
Ebola Virus GP Antibody Quantification
2
BLG-Specific Antibody and Cytokine Quantification
3
Quantification of Murine Immunoglobulin Isotypes
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Briefly, serum was isolated from control (tamoxifen) and KO (≤ 10% body weight loss) mice. The concentrations of Ig subclasses in mouse sera were determined using isotype specific antibodies with a sandwich ELISA protocol according to the manufacturer’s protocols as described previously71 (link). Monoclonal anti-mouse IgA-HRP, IgM-HRP, IgG1-HRP, IgG2b-HRP, IgG2c-HRP and IgG3-HRP were purchased from SouthernBiotech. These were used in two-fold serial dilutions starting at 10 ng/ml. Flat-bottom, 96-well plate (Nunc) was coated with capture antibody overnight at 1:250 dilution. Plates were blocked with 10% FCS in PBS buffer for 2 h and incubated with sample serum (1:10,000 for IgA and IgM and 1:20,000 for IgG1, IgG2b, IgG2c and IgG3) for 2 h at room temperature and detected with HRP-conjugated Ig subclass antibody (SouthernBiotech, 1:10,000 dilution) for 1 h at room temperature. Plates were developed with TMB substrate solution (eBioscience) and read at 450 nM using a Synergy HT Microplate Reader (BioTek).
4
BLG-Specific Antibody and Cytokine Quantification
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mMCPT-1 was quantified in serum collected 1 hour after the second challenge according to the manufacturer’s protocol (eBioscience). BLG-specific ELISAs were performed using protocols modified from ref. 7 (link). Briefly, plates were coated overnight at 4°C with 100μg/mL BLG in 100mM carbonate-bicarbonate buffer (pH 9.6). Plates were blocked for 2 hours at room temperature with 3% BSA. Samples were added in 1% BSA and incubated overnight at 4°C. Assays were standardized with BLG-specific antibodies (IgE or IgG1) purified on a CNBr-Sepharose affinity column from mice immunized with BLG+alum 42 (link). BLG-specific antibodies were detected with goat anti-mouse IgE-UNLB (Southern Biotech) and rabbit anti-goat IgG-AP (ThermoFisher) then developed with p-NPP (KPL Labs) or IgG1-HRP (Southern Biotech) and developed with TMB (Sigma).
For cytokine analysis spleens were harvested 24h post challenge from A. caccae or CMA colonized mice sensitized with BLG+CT for 5 weeks. Splenocytes were stimulated at a concentration of 2×106 cells/ml at 37°C, 10% CO2 with 10 mg/ml BLG (Sigma) in cDMEM (with 4% FCS (HyClone), 10mM HEPES (Gibco), 100U/ml Penicillin/Streptomycin (Gibco) and 55μM 2-mercaptoethanol (Gibco). Cytokine concentrations in 72 hr culture supernatants were determined by ELISA for IL-13 and IL-4 (both from Invitrogen).
For cytokine analysis spleens were harvested 24h post challenge from A. caccae or CMA colonized mice sensitized with BLG+CT for 5 weeks. Splenocytes were stimulated at a concentration of 2×106 cells/ml at 37°C, 10% CO2 with 10 mg/ml BLG (Sigma) in cDMEM (with 4% FCS (HyClone), 10mM HEPES (Gibco), 100U/ml Penicillin/Streptomycin (Gibco) and 55μM 2-mercaptoethanol (Gibco). Cytokine concentrations in 72 hr culture supernatants were determined by ELISA for IL-13 and IL-4 (both from Invitrogen).
5
ELISA Assay for S-Protein Antibody Titers
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S-protein-specific serum antibody titers were measured using ELISA. Corning ELISA plates were incubated with 10 µg/mL of the recombinant S protein purified in our lab in phosphate-buffered saline (PBS) overnight at 4 °C. After three washes, the plates were blocked with 1 × ELISAPOT Diluent (Invitrogen, cat. 00-4202-56, Waltham, MA, USA) for 2 h at 25 °C. Following another washing, the plates were incubated overnight using mouse serum at 4 °C and diluted with PBST. Following a set of washings, the plates were incubated with a 1:5000 dilution of goat anti-mouse IgG (H + L)-BIOT, IgG1-HRP, IgG2a-HRP or IgG3-HRP (Southern Biotech, cat. 1036-08, 1071-05, 1081-05 and 1101-05) for 1 h at room temperature. For IgG (H + L)-BIOT, following three washes, the plates were incubated with streptavidin-HRP (Solarbio, cat. SE068, Beijing, China) for 30 min at 25 °C. Finally, after washing the plates, the reaction was revealed using TMB 1-Component Peroxidase Substrate (Invitrogen, cat. 00-4201-56) and impeded using a 2 M HCl solution. The absorbance at 450 nm was determined within 30 min using a Synergy HTX instrument (BioTek Instruments, Highland Park, VT, USA).
6
ELISA Antibody Isotype Detection
7
Ebola Glycoprotein Antibody Assays
8
Mouse IgG and Isotype Detection
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Goat anti-mouse total IgGFc-HRP (Jackson ImmunoResearch), goat anti-mouse IgM-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG2c-HRP or IgG3-HRP (Southern Biotechnology Associates) were used as secondary antibodies (dilutions 1:1000). Antibody was detected using ABTS substrate (Fisher Scientific, NH, USA).
9
Serum Antibody Titers against ID97 Antigen
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Serum titers against ID97 antigen were evaluated by antibody-capture ELISA. Briefly, Corning high-binding 384-well plates (VWR International, Radnor, PA, USA) were coated overnight at 4 °C with 2 µg/mL ID97 in coating buffer (eBioscience, San Diego, CA, USA), then washed in phosphate-buffered saline (PBS)-Tween 20. Serially diluted serum samples were incubated for 1 h followed by either anti-mouse IgG (H + L)-HRP, IgG1-HRP, or IgG2c-HRP (SouthernBiotech, Birmingham, AL, USA); and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was applied as per the manufacturer’s instructions. Plates were analyzed at 450 nm using an ELx808 Absorbance Reader (Bio-Tek Instruments, Winooski, VT, USA), and endpoints were set as the minimum dilution at which values were lesser than or equal to an OD of 0.5.
10
Chitosan-Based Nanoparticle Synthesis Protocol
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Chitosan (medium molecular weight (50–190
kDa); degree of deacetylation of 75–85%), IGEPAL CO-520, sodium
chloride, calcium chloride dihydrate, bovine serum albumin-FITC (BSA-FITC),
albumin from white chicken egg (OVA; purity, ≥98%), glycerol,
and goat anti-mouse IgG-HRP were all purchased from Sigma. Sodium
alginate was purchased from the Melbourne Food Depot and cyclohexane
was obtained from Ajax Finechem. Disodium hydrogen orthophosphate
was from AnalaR. Soybean trypsin inhibitor was purchased from FUJIFILM
Chemical. TMB single solution was bought from Life Technologies and
goat anti-mouse IgA, IgG2a, and IgG1-HRP were all purchased from Southern
Biotech. Trypsin–EDTA (0.25%), PBS buffer (37 mmol/L NaCl,
2.7 mmol/L KCl, 8.1 mmol/L Na2HPO4, and 1.47
mmol/L KH2PO4; pH 7.4) and DMEM medium were
obtained from Life Technologies Corporation (Australia).
kDa); degree of deacetylation of 75–85%), IGEPAL CO-520, sodium
chloride, calcium chloride dihydrate, bovine serum albumin-FITC (BSA-FITC),
albumin from white chicken egg (OVA; purity, ≥98%), glycerol,
and goat anti-mouse IgG-HRP were all purchased from Sigma. Sodium
alginate was purchased from the Melbourne Food Depot and cyclohexane
was obtained from Ajax Finechem. Disodium hydrogen orthophosphate
was from AnalaR. Soybean trypsin inhibitor was purchased from FUJIFILM
Chemical. TMB single solution was bought from Life Technologies and
goat anti-mouse IgA, IgG2a, and IgG1-HRP were all purchased from Southern
Biotech. Trypsin–EDTA (0.25%), PBS buffer (37 mmol/L NaCl,
2.7 mmol/L KCl, 8.1 mmol/L Na2HPO4, and 1.47
mmol/L KH2PO4; pH 7.4) and DMEM medium were
obtained from Life Technologies Corporation (Australia).
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