ELISA was performed as previously described [45 (link)]. The wells of 96-well microplates were coated with NP-His protein (0.5 μg/mL). Then, the microplates were washed with PBST (PBS containing 0.1% Tween 20), followed by blocking with 1% bovine serum albumin (BSA). Serial dilutions of the indicated NP antibodies were added to the wells and incubated at room temperature (RT) for 1 h. After washing with PBST, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was incubated in the wells at RT for 1 h. Finally, the plates were incubated with chromogenic substrate 3,3′,5,5,’-tetramethylbenzidine (TMB; Sigma-Aldrich, St. Louis, MO, USA). The reactions were stopped with 3-N HCl, and the optical density was measured using a microplate reader at 450 nm.
Horseradish peroxidase hrp conjugated anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase (HRP)-conjugated anti-mouse IgG is a laboratory reagent used for immunodetection. It is an antibody-enzyme conjugate that specifically binds to mouse immunoglobulin G (IgG) and can be used to detect the presence of mouse IgG in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
C digit, by LI COR (1 mentions)
Anti flag f1804, by Merck Group (1 mentions)
Immunoplates, by SPL Life Sciences (1 mentions)
Elisa plate reader, by Molecular Devices (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Elisa plate, by Corning (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions)
Nupage tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate enhanced chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Casein blocking buffer, by Merck Group (1 mentions)
Emax microplate reader, by Molecular Devices (1 mentions)
Tmb 3 3 5 5 tetramethylbenzidine, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
High binding 96 half well microplates, by Corning (1 mentions)
Schneider s drosophila medium, by Thermo Fisher Scientific (1 mentions)
Immobilon western, by Merck Group (1 mentions)
11 protocols using horseradish peroxidase hrp conjugated anti mouse igg
1
Quantification of NP-Specific Antibodies by ELISA
2
Quantitative Micro-Western Blot Analysis
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Micro-Western blot was performed as described in a previous report [22 ]. The oocyte numbers used in each experiment are indicated in the figure
legends. The antibodies used were: anti-CCNB1 (ADI-KAM-CC195-E, mouse IgG monoclonal antibody; Enzo Life Sciences, NY, USA), anti-Cdc2 (sc-54, mouse IgG monoclonal antibody; Santa Cruz, TX,
USA), and anti-Flag (F1804, mouse IgG monoclonal antibody; Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, PA, USA) was used as
secondary antibody. Signals were detected using ImmunoStar LD (Wako, Tokyo, Japan) and C-Digit (Li-Cor; Lincoln, NE, USA), as instructed by manufacturers. The relative intensity of the
endogenous CCNB1 was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
legends. The antibodies used were: anti-CCNB1 (ADI-KAM-CC195-E, mouse IgG monoclonal antibody; Enzo Life Sciences, NY, USA), anti-Cdc2 (sc-54, mouse IgG monoclonal antibody; Santa Cruz, TX,
USA), and anti-Flag (F1804, mouse IgG monoclonal antibody; Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, PA, USA) was used as
secondary antibody. Signals were detected using ImmunoStar LD (Wako, Tokyo, Japan) and C-Digit (Li-Cor; Lincoln, NE, USA), as instructed by manufacturers. The relative intensity of the
endogenous CCNB1 was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
ELISA-based IgG antibody detection
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Levels of IgG antibodies against IAV and SARS-CoV-2 were measured in sera of infected mice collected at day 10 postinfection. Briefly, 96-well Immuno plates (SPL Life Science, Gyeonggi-do, South Korea) were coated overnight at 4°C with 500 TCID50/well of inactivated IAV or SARS-CoV-2 in a carbonate solution. The plates were blocked with 5% skim milk in PBS for 2 h at 37°C and were washed with 0.05% Tween 20 in PBS. The plates were then incubated with the diluted sera (1:50 to 1:6,400) for 2 h at 37°C followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:3000) (Jackson ImmunoResearch Laboratories, Inc., PA, USA) for 1 h at 37°C. The reactions were developed using o-phenylenediamine dihydrochloride for 30 min and stopped with 3 M HCl. The absorbance of the samples was measured at 450 nm using an ELISA plate reader (Molecular Devices, San Jose, USA).
4
Quantifying Sporozoite-specific Antibodies
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Enzyme-linked immunosorbent assay (ELISA) plates (Corning, Inc.) were coated overnight at 4°C by adding 1 × 104PyΔfabb/f sporozoite lysate diluted in 100 μl NaHCO3 buffer (pH 9,6) per well. Plates were washed three times with PBS-T (0.05% Tween 20 in 1 × PBS) prior to blocking for 2 h in blocking buffer (1% BSA in PBS-T). Next, sera was diluted in blocking buffer at 1:100 for sporozoite lysate per well. Plates were incubated for 3 h at room temperature before washing as described above. Next, 100 μl of a 1:5000 dilution of horseradish peroxidase (HRP) conjugated anti-mouse IgG (Jackson Immuno-Research) was added and incubated for an additional 1 h at room temperature. Finally, plates were washed again and 100 μl of TMB Substrate solution (Thermo Scientific) was added for 5 min. The reaction was stopped by addition of 50 μl of 0.5 N sulfuric acid prior to measurement of absorbance at 450 nm using a Multiskan FC (Thermo Scientific) microplate reader.
5
Testicular Protein Extraction and Western Blot
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Testes (40 pairs/sample) were dissected in Schneider’s media at room temperature within 30 minutes, the media was removed and the samples were frozen at -80°C until use. After thawing, testes were then lysed in 200uL of 2X Laemmli Sample Buffer + βME (BioRad, 161–0737). Samples were separated on a NuPAGE Tris-Acetate gel (3–8%, 1.5mm, Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore) using NuPAGE transfer buffer (Invitrogen) without added methanol. Membranes were blocked in 1X TBST (0.1% Tween-20) containing 5% nonfat milk, followed by incubation with primary antibodies diluted in 1X TBST containing 5% nonfat milk. Membranes were washed with 1X TBST, followed by incubation with secondary antibodies diluted in 1X TBST containing 5% nonfat milk. After washing with 1X TBST, detection was performed using the Pierce® ECL Western Blotting Substrate enhanced chemiluminescence system (Thermo Scientific). Primary antibodies used were anti–α-tubulin (1:2,000; mouse, monoclonal, clone DM1a; Sigma-Aldrich) and anti-FLAG (1:2,500; mouse, monoclonal, M2, Sigma-Aldrich). The secondary antibody was horseradish peroxidase (HRP) conjugated anti-mouse IgG (1:10,000; Jackson ImmunoResearch Laboratories).
6
ASFV Antibody Detection Assay
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Anti-ASFV porcine serum was collected 48 days post-inoculation (dpi) with ASFV (Estonia 2014). Microtitre plates (Nunc-Immunoplate Maxisorp, Roskilde, Denmark) were coated with polyclonal porcine anti-ASFV (Estonia 2014) serum diluted 1:5000 in a carbonate/bicarbonate buffer, pH 9.6 overnight at 4°C. After blocking in casein blocking buffer (Sigma-Aldrich, United States), ASF viruses (Table 1 ) were added. Concentrated and irradiated ASFV Lisbon/61 was used as an antigen for hybridoma screening. Hybridoma culture supernatants were then added. After incubation, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:2000, Jackson ImmunoResearch Laboratories, West Grove, PA, United States) was added, followed by-3,3′,5,5′-Tetramethylbenzidine (TMB, Pierce Biotechnology, Inc. Rockford, Illinois, United States). After stopping, optical density (OD) was measured at 450 nm using an Emax microplate reader (Molecular Devices, San Jose, CA, United States). Each incubation step was 60 min at 37°C with gentle shaking, followed by five washes (PBS with 0.1% Tween 20).
7
Evaluation of Anti-Mtb Antibodies by ELISA
8
Transient Overexpression of NAS-6 in S2 Cells
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S2 cells were suspended in Schneider's Drosophila medium (Gibco, 21720-024) with 5% fetal bovine serum (SAFC Biosciences) and penicillin (100 units/mL)/streptomycin (100 µg/mL) (Fujifilm, 168-23191) and seeded on a 6 cm sterile dish at 6.0 × 106 cells per dish. Actin-Gal4 (0.6 µg) and 1.2 µg of UAS::Ppa-nas-6::HA or UAS::Cel-nas-6::HA were transfected into the cells using Effectene Transfection Reagent (QIAGEN, 301427). The cells were incubated for 3 days, and the supernatant and whole-cell lysates were separately prepared for SDS–PAGE and western blotting. To concentrate NAS-6 in the supernatant, 15 µL/sample of Pierce anti-HA magnetic beads (Thermo Fischer, 88836) was used. Western blotting was performed using the following primary antibodies: mouse anti-HA (Sigma, 11583816001; 1:500) and mouse antihistone (Active Motif, 39064; 1:2000) and a secondary antibody, horseradish peroxidase (HRP)-conjugated antimouse IgG (Jackson ImmunoResearch, 715-035-151; 1:50000). Chemiluminescence of HRP substrate (Immobilon Western, Millipore, WBKLS00500) was detected using ChemiDoc MP Imaging System (Bio-Rad, 17001402JA).
9
SARS-CoV-2 S RBD Antibody Detection
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S protein receptor-binding domain (S RBD)-specific antibody titers in serum and BALF were determined using ELISAs. The recombinant His-tagged SARS-CoV-2 S RBD protein (aa 319–541) was produced in Expi293F cells using a transient expression system (Thermo Fisher) according to the manufacturer's instructions and purified using Nuvia™ IMAC Resin (Bio-Rad). NUNC-MaxiSorp™ 96-well plates (Thermo Scientific) were coated with 1 μg/ml of recombinant SARS-CoV-2 S RBD protein overnight at 4 °C and blocked with 5% fetal bovine serum (FBS) in DBPS for 1 h at RT. Serially diluted samples were added to wells and incubated overnight at 4 °C. After washing four times with ELISA washing buffer (DPBS containing 0.05% Tween-20), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) or anti-mouse IgA (Southern Biotech, Birmingham, AL, USA) antibodies were used to detect S RBD-specific antibodies in the samples. Plates were incubated for 1 h at RT and washed four times with ELISA washing buffer. TMB (eBioscience, San Diego, CA, USA) was used as the substrate, and relative titers were calculated based on the absorbance at 450 nm using a SpectraMax® ABS Plus Absorbance Microplate Reader (Molecular Devices, San Jose, CA, USA).
10
HIV/SIV Env-specific IgG ELISA Assay
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