Electrophoresis was performed using 12–15% SDS-PAGE to characterize the molecular weights of the different recombinant proteins. To confirm the immunological characteristics of the purified proteins, serum samples were diluted with PBS containing Tween 20 (PBST), and samples were collected from immunized mice in the rNspA and HBc-N144-NspA groups, respectively, bound to the anti-His-tag antibody. Briefly, purified proteins were transferred to a Polyvinylidene Fluoride membrane (PVDF). The PVDF membrane was blocked with blocking buffer and incubated with the anti-His antibody.19 (link) Protein bands were visualized using horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Sigma, St. Louis, MO, USA) and using chemiluminescence kit for detection (GE Healthcare, Chicago, IL, USA)
Hrp conjugated rabbit anti mouse igg
Manufactured by Merck Group
Sourced in United States
HRP-conjugated rabbit anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is conjugated with horseradish peroxidase (HRP). This product can be used in various immunoassay techniques, such as ELISA, Western blotting, and immunohistochemistry, to detect and quantify mouse IgG in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
O phenylenediamine, by Merck Group (2 mentions)
Duoset elisa development kit, by R&D Systems (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ecl prime wb detection reagent, by GE Healthcare (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Polystyrene plates, by Thermo Fisher Scientific (1 mentions)
Ecl chemiluminescent detection system, by Cytiva (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Hrp conjugated goat anti human igg, by Merck Group (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
Tyramine hydrochloride, by Merck Group (1 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated streptavidin, by Merck Group (1 mentions)
Hemin, by Merck Group (1 mentions)
Mops running buffer, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose 4b, by Merck Group (1 mentions)
Biotinylated goat anti human igg fc, by Thermo Fisher Scientific (1 mentions)
Biotinylated malii, by Vector Laboratories (1 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
15 protocols using hrp conjugated rabbit anti mouse igg
1
Characterization of Recombinant Proteins
2
Quantifying FH, C3, and C5a Levels by ELISA
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ELISAs for FH, C3, and C5a were performed as previously described, with modifications [6 (link), 14 (link)]. Serum FH was detected using mouse anti-human FH CCP1 IgG (Quidel), a region of FH not contained within the fusion protein, followed by HRP-conjugated rabbit anti-mouse IgG (Sigma). Total C3 was detected using chicken anti-human C3 IgY (Millipore), followed by HRP-conjugated goat anti-chicken IgG (Invitrogen); C5a was detected using the DuoSet ELISA Development kit, per manufacturer’s instructions (R&D systems). Plates were developed with TMB substrate, stopped with 2N H2SO4, and then read at 450nm. Purified FH (CompTech), C3 (CompTech) or C5a (R&D systems) were used as standards for their respective ELISAs.
3
CCHF Virus Protein Detection in SW13 Cells
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SW13 cell monolayers were infected with CCHF virus strain IbAr10200 at a multiplicity of infection (MOI) of approximately 0.01, and incubated at 37°C in Leibovitz's L-15 medium containing 2% foetal bovine serum. 48 hours post-infection, the medium was removed and the cells were treated with Laemmli buffer supplemented to contain 10% sodium dodecyl sulfate (SDS) (Sigma). The resultant mixture was collected into vials and heat treated at 90°C for 10 minutes before use in Western Blot analysis. Uninfected SW13 monolayers were treated similarly for use as a negative control.
Lysates from CCHFv-infected or uninfected SW13 cells were subjected to SDS-PAGE on a 4–12% Bis-Tris gel (Life Technologies) and transferred to a PVDF membrane. After blocking in 5% milk protein, membranes were incubated with mouse serum for 2 hours, washed 6 times with PBS containing 0.05% NP40, incubated for 1 hour with HRP-conjugated rabbit anti-mouse IgG (Sigma) or goat anti-mouse IgG/A/M (AbD Serotec) and washed as before. All antibody dilutions were made in PBS containing 0.05% NP40 and 5% milk protein. Bound antibody was detected with ECL-Prime WB detection reagent (GE Life Sciences, UK) according to the manufacturer's directions and visualised on a ChemiDoc system (BioRad, UK). Molecular weights were calculated by comparison with markers of known molecular weight using QuantityOne software.
Lysates from CCHFv-infected or uninfected SW13 cells were subjected to SDS-PAGE on a 4–12% Bis-Tris gel (Life Technologies) and transferred to a PVDF membrane. After blocking in 5% milk protein, membranes were incubated with mouse serum for 2 hours, washed 6 times with PBS containing 0.05% NP40, incubated for 1 hour with HRP-conjugated rabbit anti-mouse IgG (Sigma) or goat anti-mouse IgG/A/M (AbD Serotec) and washed as before. All antibody dilutions were made in PBS containing 0.05% NP40 and 5% milk protein. Bound antibody was detected with ECL-Prime WB detection reagent (GE Life Sciences, UK) according to the manufacturer's directions and visualised on a ChemiDoc system (BioRad, UK). Molecular weights were calculated by comparison with markers of known molecular weight using QuantityOne software.
4
Enzyme-Linked Immunosorbent Assay for Brucella Antibodies
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Wells in polystyrene plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with rOmp19, rOmp25, or rOmp31 at 5.0 μg/mL in bicarbonate buffer, pH 9.6 and incubated at 4°C overnight. The plate was then washed 3 times sequentially with PBS and PBS supplemented with Tween-20 (PBS-T). Antisera dilutions were prepared in PBS-T (1:100–1:12,800) which were added to 8 wells coated with each of the proteins; the plate was incubated at 37°C for 1 h. After washing, (HRP)-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) diluted with PBS-T was added to each well. The plate was incubated for another 1 h at 37°C, washed 3 times, and then developed with O-phenylenediamine (Sigma-Aldrich, St. Louis, USA). The reactions were stopped after 3-5 min by adding an equal amount of 2 M sulfuric acid to each well. The absorbance was measured at 490 nm using a plate reader (Bio-Rad 680, Redmond, WA, USA). The cutoff value for the assay was calculated as the mean optical density (OD) plus 3 standard deviations for 18 negative control sera obtained from mice before immunization.
5
Western Blot Detection of Properdin
6
Antibody Titration in Vaccinated Cattle
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Total antibody titres in vaccinated cattle were detected with a SAT/ZIM/7/83-specific liquid-phase blocking enzyme-linked immunosorbent assay (ELISA) (LPBE) [15 (link),35 (link)]. Antibodies were measured in serum collected on days 0, 2, 4, 7, 9, 11, 14, and 21 pv and thereafter every fortnight until 162 dpv. Serum samples from 0–11 dpc were also tested.
The reactivity of antibodies in 162 dpv sera were also tested in triplicate wells against the vKNPS2aSAT2 and vKNPS2bSAT2 viruses in a competition ELISA format, as described previously by Opperman et al. (2014) [31 (link)]. Briefly, flat-bottom 96-well plates were coated with rabbit SAT2 antiserum in 50 mM carbonate buffer (pH 9.6). SDG-purified (60 ng/well) vKNPS2aSAT2, vKNPS2bSAT2 and parental viruses were applied. Sera (162 dpv) was diluted (1:6) in blocking buffer and antibodies detected via competition with monoclonal antibody GD12 (1:40) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Sigma-Aldrich) (1:7000). Following color development, the reaction was stopped and the (OD450) values measured. The maximum OD450 value was calculated from the negative control and the average readings of two ELISAs were used.
The reactivity of antibodies in 162 dpv sera were also tested in triplicate wells against the vKNPS2aSAT2 and vKNPS2bSAT2 viruses in a competition ELISA format, as described previously by Opperman et al. (2014) [31 (link)]. Briefly, flat-bottom 96-well plates were coated with rabbit SAT2 antiserum in 50 mM carbonate buffer (pH 9.6). SDG-purified (60 ng/well) vKNPS2aSAT2, vKNPS2bSAT2 and parental viruses were applied. Sera (162 dpv) was diluted (1:6) in blocking buffer and antibodies detected via competition with monoclonal antibody GD12 (1:40) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Sigma-Aldrich) (1:7000). Following color development, the reaction was stopped and the (OD450) values measured. The maximum OD450 value was calculated from the negative control and the average readings of two ELISAs were used.
7
Antibody Binding Assay for rOmps
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The polystyrene plate wells were coated with chimeric proteins, and mice anti-rOmps19, 25, and 31 sera were diluted as described above. In addition, antibodies bound to the plate were detected with HRP-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). The cutoff value for the assay was calculated as the mean-specific OD plus three standard deviations (SDs) for five sera from nonimmunized mice assayed at a 1:100 dilution.
8
Antibody Titers Determination by ELISA
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Antibody titres were determined by ELISA as described previously [32 (link)]. Briefly, ELISA plates were coated overnight at room temperature with 10 μg/ml of peptides or 5 μg/ml of DENV recombinant E proteins (Hawaii biotech, USA). Wells were blocked with 50 μl of purified IgG diluted to 20 μg/ml. For individual or pooled mice sera, serial half-log dilutions were prepared and 50 μl were added to each well. The plates were incubated for 1 hour and the sera or purified IgG dilutions were removed and the wells were washed twice. An aliquot of 50 μl (1:1000 dilution) of HRP-conjugated goat anti-human IgG (Sigma-Aldrich, MO, USA) or HRP-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, MO, USA) was added. The optical density (OD) of individual wells was read by an iMark Microplate Reader (BioRad Laboratories, Hercules, CA, USA) at a wavelength of 450nm. Antibody titres were expressed as the reciprocal of the logarithm of that dilution of serum that gave an OD four times above that obtained in wells with pre-immune control sera.
9
Monoclonal Antibody Specificity Assay
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The specificity of produced monoclonal antibodies was tested by IgG ELISA against recombinant LigA (LK90) protein and heat killed antigens prepared from MACS13 (link) of different leptospiral serovars, and heat killed antigens from bacterial pathogens other than Leptospira. Two micrograms of each representative proteins per well were coated on a flat-bottom polystyrene microtiter plate at 4 °C overnight, using carbonate coating buffer (pH 9.6), followed by blocking with 4% non-fat dry milk. Purified monoclonal antibodies (1:100) in triplicate were added and incubated for 1 h at 37 °C. Bound IgG was detected using HRP-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) at a dilution of 1:8,000. Plates were developed with o–phenylenediamine (Sigma-Aldrich, St. Louis, MO). The reaction was stopped with the addition of 50 µl of 1 N H2SO4, and the optical density was measured at 490 nm (Bio-Rad, USA).
10
Purification and Characterization of Sn-Fc Chimeras
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Sn-HRP-Fc DNA constructs were synthesized by GenScript (GenScript HK Limited). The Sn-Fc chimeras (Crocker et al. 1999 (link)) and Sf9 cells were from laboratory stocks. Sf-900 II serum free medium, FetalClone II serum, Dynabeads® MyOne™ Streptavidin C1 magnetic beads, sulfo-NHS-SS-biotin, NuPAGE® sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, MOPS running buffer, LDS sample buffer, sample reducing agent, and biotinylated goat anti-human IgG Fc were from Thermo Fisher Scientific (Paisley, UK). Tyramine hydrochloride, hemin, protein A-Sepharose®4B, FITC-conjugated goat anti-human IgG Fc, HRP-conjugated rabbit anti-mouse IgG and HRP-conjugated streptavidin were from Sigma (Dorset, UK). HRP-conjugated goat anti-human IgG Fc was from Abcam (Cambridge, UK). Mouse anti-human glycophorin A (Clone: BRIC 163) was from IBGRL Research Products (Bristol, UK). Biotinylated MAL II was from Vector Laboratories (Peterborough, UK). Complete™, Mini, EDTA-free protease inhibitor cocktail was from Roche (Boehringer, UK). Human erythrocytes were collected from volunteer donors under human subject protocols approved by the local ethics committee at the University of Dundee.
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