The amount of infectious JEV in tissues and culture media was measured by FFA. Briefly, the FFA was performed by incubating 4-fold serially diluted virus (from 1:4 to 1:64) with Vero cells (around 80% confluent) in 24-well plates. After 1 h at 37 °C, the wells were overlaid with 0.5 ml 0.8% methylcellulose (Thermo Fisher Scientific, Inc., Waltham, MA) in Opti-MEM Media (Invitrogen Corp., Carlsbad, CA) containing 2% FBS. Following a 3-day incubation, the methylcellulose overlay was removed, and the cells in each well were washed with PBS, followed by fixation with cold MeOH. The cells were blocked, permeabilized with 10% goat serum containing 0.5% TritonX100 for 1 h at room temperature, followed by washing twice with PBS. Subsequently, the cells were stained intracellularly with mAb specific for JEV NS1 protein (Abcam, Cambridge, UK) for 90 min, followed by staining with HRP-conjugated goat anti-mouse IgG (H + L) (Southern Biotech). The focus-forming spots were visualized using WEST-ZOL Plus (iNtRON, Daejeon, Korea) in Fusion FX7 (Vilber Lourmat, Collegien, France), according to the manufacturer’s protocol. Photographs were taken on the same day as staining. The ffu/culture media or tissue was calculated by counting focus-forming spots.
Hrp conjugated goat anti mouse igg h l
Manufactured by Southern Biotech
Sourced in United Kingdom
HRP-conjugated goat anti-mouse IgG (H + L) is a secondary antibody used in various immunoassay techniques. It is produced by immunizing goats with mouse immunoglobulins and conjugating the antibodies with horseradish peroxidase (HRP).
Automatically generated - may contain errors
Lab products found in correlation
Opti mem media, by Thermo Fisher Scientific (1 mentions)
Fusion fx7, by Vilber (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Cytometric bead array, by BioLegend (1 mentions)
Igg2a, by Southern Biotech (1 mentions)
H3n2 ha, by Sino Biological (1 mentions)
H1n1 ha, by Sino Biological (1 mentions)
H5n1 ha, by Sino Biological (1 mentions)
2 n h2so4 solution, by Thermo Fisher Scientific (1 mentions)
1 step ultra tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Synergy h4 microplate reader, by Agilent Technologies (1 mentions)
4 protocols using hrp conjugated goat anti mouse igg h l
1
Quantifying Japanese Encephalitis Virus by FFA
2
Murine Autoantibody Detection by ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detection of murine autoantibodies by ELISA, microtiter plates were coated with 10 μg/mL nucleosomes isolated from apoptotic cells as described previously (73 (link)), 50 μg/mL cardiolipin (MilliporeSigma, C1649), and 10 U/mL Sm/RNP (GenWay Biotech, GWB-A1CECE) or precoated with 20 μg/mL poly-l -lysine (MilliporeSigma, P2658) before adding 20 μg/mL calf thymus DNA (MilliporeSigma, D3664). Sera were 1:50 or 1:25 diluted in PBS/2% FBS (Thermo Fisher Scientific, 26140079). Bound IgG was detected with HRP-conjugated goat anti-mouse IgG(H+L) (Southern Biotech, 1031-05) at 1:4,000 dilution, followed by substrate solution (Roche, 11112422001). Results were expressed as fold-changes relative to a designated positive sample. IFN-α level in peritoneal lavages and cell culture supernatants was quantified by cytometric bead array (BioLegend, 740633).
3
ELISA for Flu Antigen Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The titers of serum IgG, IgG1, and IgG2a were measured
using an enzyme-linked
immunosorbent assay (ELISA). Briefly, 1 μg/mL of each flu antigen,
including H1N1 HrHA (derived from A/Puerto Rico/8/1934), H1N1 HA (A/California/04/2009),
H3N2 HA (A/Aichi/2/1968) (SinoBiological), H5N1 HA (A/Vietnam/1194/2004)
(SinoBiological), and tZE-4M2e, in PBS was coated onto Maxisorp 96
well immune assay plates during overnight incubation at 25 °C.
For the evaluation of off-target immune responses, Maxisorp 96-well
plates were coated with 1 μg/mL of tGCN4, tZR, or ZE-mCherry.
The next day, each well was washed three times with PBS containing
0.1% Tween-20 and blocked with 1% BSA-supplemented PBS for 2 h at
25 °C. Each well was incubated with serially diluted sera for
1 h at 25 °C and washed three times. Then, a 1:5000 dilution
of HRP-conjugated goat antimouse IgG (H+L) (SouthernBiotech), IgG1
(SouthernBiotech), or IgG2a (SouthernBiotech) secondary antibodies
was added to each well, incubated for 1 h at 25 °C, and washed
three times. Each well was developed with 1-Step Ultra TMB substrate
solution for 15 min and stopped with 2 N H2SO4 solution. Absorbance values at 450 nm were measured on a BioTek
Synergy H4 Micro plate reader with the correction wavelength set to
540 nm.
using an enzyme-linked
immunosorbent assay (ELISA). Briefly, 1 μg/mL of each flu antigen,
including H1N1 HrHA (derived from A/Puerto Rico/8/1934), H1N1 HA (A/California/04/2009),
H3N2 HA (A/Aichi/2/1968) (SinoBiological), H5N1 HA (A/Vietnam/1194/2004)
(SinoBiological), and tZE-4M2e, in PBS was coated onto Maxisorp 96
well immune assay plates during overnight incubation at 25 °C.
For the evaluation of off-target immune responses, Maxisorp 96-well
plates were coated with 1 μg/mL of tGCN4, tZR, or ZE-mCherry.
The next day, each well was washed three times with PBS containing
0.1% Tween-20 and blocked with 1% BSA-supplemented PBS for 2 h at
25 °C. Each well was incubated with serially diluted sera for
1 h at 25 °C and washed three times. Then, a 1:5000 dilution
of HRP-conjugated goat antimouse IgG (H+L) (SouthernBiotech), IgG1
(SouthernBiotech), or IgG2a (SouthernBiotech) secondary antibodies
was added to each well, incubated for 1 h at 25 °C, and washed
three times. Each well was developed with 1-Step Ultra TMB substrate
solution for 15 min and stopped with 2 N H2SO4 solution. Absorbance values at 450 nm were measured on a BioTek
Synergy H4 Micro plate reader with the correction wavelength set to
540 nm.
4
ELISA-based Quantification of Antigen-Antibody Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OVA SAPNs
and HrHA SAPNs with 10-fold serial dilutions (10–4–1 ng/μL and 10–6–1 ng/μL,
respectively) were coated onto Maxisorp 96 well immune assay plates
(Nunc) and incubated overnight at 25 °C. The next day, the coated
plates were washed three times with PBS containing 0.1% Tween-20 and
each well was blocked with 1% bovine serum albumin (BSA) in PBS for
1 h at 25 °C. Then, the plates were washed three times and incubated
with HRP-conjugated rabbit anti-OVA antibodies (Thermo Fisher Scientific)
diluted at 1:3,000 for 1 h at 25 °C. For anti-HrHA and anti-4M2e
antibody binding assay, the plates were incubated with anti-HrHA and
anti-4M2e serum antibodies, which were harvested from mice administered
with 2 doses of 20 μg of HrHA-tGCN4 and 20 μg of tZE-4M2e,
diluted at 1:5000 and 1:2500, respectively, for 1 h at 25 °C.
Afterward, plates incubated with the anti-HrHA and anti-4M2e serum
antibodies were washed and incubated with a 1:5000 dilution of HRP-conjugated
goat antimouse IgG (H+L) (SouthernBiotech) for 1 h at 25 °C.
Each well was washed three times and revealed with 1-Step Ultra TMB
substrate solution (Thermo Fisher Scientific) for 20 min. The enzymatic
activity of HRP was stopped by adding 2 N H2SO4 solution (Thermo Fisher Scientific). Absorbance at 450 nm was measured
on a BioTek Synergy H4 Micro plate reader with the correction wavelength
set to 540 nm.
and HrHA SAPNs with 10-fold serial dilutions (10–4–1 ng/μL and 10–6–1 ng/μL,
respectively) were coated onto Maxisorp 96 well immune assay plates
(Nunc) and incubated overnight at 25 °C. The next day, the coated
plates were washed three times with PBS containing 0.1% Tween-20 and
each well was blocked with 1% bovine serum albumin (BSA) in PBS for
1 h at 25 °C. Then, the plates were washed three times and incubated
with HRP-conjugated rabbit anti-OVA antibodies (Thermo Fisher Scientific)
diluted at 1:3,000 for 1 h at 25 °C. For anti-HrHA and anti-4M2e
antibody binding assay, the plates were incubated with anti-HrHA and
anti-4M2e serum antibodies, which were harvested from mice administered
with 2 doses of 20 μg of HrHA-tGCN4 and 20 μg of tZE-4M2e,
diluted at 1:5000 and 1:2500, respectively, for 1 h at 25 °C.
Afterward, plates incubated with the anti-HrHA and anti-4M2e serum
antibodies were washed and incubated with a 1:5000 dilution of HRP-conjugated
goat antimouse IgG (H+L) (SouthernBiotech) for 1 h at 25 °C.
Each well was washed three times and revealed with 1-Step Ultra TMB
substrate solution (Thermo Fisher Scientific) for 20 min. The enzymatic
activity of HRP was stopped by adding 2 N H2SO4 solution (Thermo Fisher Scientific). Absorbance at 450 nm was measured
on a BioTek Synergy H4 Micro plate reader with the correction wavelength
set to 540 nm.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!