Levels of neutralizing antibodies bound by prefusion and postfusion F structures were determined with a competitive ELISA. Cotton rat serum was serially diluted in 96-well microtiter plates and 5 µg mL−1 prefusion F (BV2129) or postfusion F (BV2128) added for 1 h at 37 °C followed by addition of RSV/A (TCID50 = 200–300) for 2 h at 37 °C. Following incubation, 2.5 × 104 HEp-2 cells were added to the wells and the plates incubated at 37 °C and 5% CO2 for 4 days. After incubation, the cultures were washed and air dried. Virus replication was detected with the mouse mAb RSV5H5 anti-RSV M2-1 (ab94805, Abcam, Cambridge, MA, USA) followed by addition of HRP-conjugated goat anti-mouse IgG secondary antibody (catalog number 1030-05, Southern Biotech, Birmingham, AL, USA). TMB substrate was added and the plates read at OD 450 nm with a plate reader. Percent neutralization was calculated compared to the virus control cultures (no serum).
Hrp conjugated goat anti mouse igg secondary antibody
Manufactured by Southern Biotech
Sourced in United States
The HRP-conjugated goat anti-mouse IgG secondary antibody is a laboratory tool used for the detection and quantification of mouse immunoglobulin G (IgG) in various immunoassays. The antibody is conjugated with horseradish peroxidase (HRP), which enables the visualization and amplification of the target signal.
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Lab products found in correlation
8 protocols using hrp conjugated goat anti mouse igg secondary antibody
1
RSV Neutralizing Antibody Assay
2
Coronavirus Antigen Binding ELISA Assay
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For coronavirus antigen-binding assays, 384-well ELISA plates (Costar #3700) were coated with 2 μg/ml antigens in 0.1M sodium bicarbonate overnight at 4°C. Plates were then washed 1X and blocked for 2 h at room temperature with SuperBlock (1X phosphate buffered saline (PBS) containing 4% (w/v) whey protein 15% normal goat serum/0.5% Tween-20/0.05% sodium azide). Mouse serum samples were collected at baseline before prime, two weeks post prime, four weeks post prime, two weeks post boost, and two weeks post the second boost. Mouse serum samples were added at 1:30 dilution in SuperBlock and diluted 3-fold through 12 dilution spots to generate binding curves. Diluted serum samples were bound to coated plates in SuperBlock for 1h at room temperature. Plates were then washed 2X and a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (SouthernBiotech 1030–05) was added in SuperBlock at a 1:16,000 dilution. Secondary antibody was bound for 1h and then washed 4X and detected with 20μL SureBlue Reserve (KPL 53–00-03) for 15 min. Colorimetric reactions were stopped by adding 20μL of 1% HCL stop solution. Plates were read at 450nm and area under the curve (AUC) was calculated from the serially diluted mouse serum samples.
3
CHIKV E2 Antibody Titer Assay
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For determining
E2-specific antibody titers, 96-well ELISA plates (Nunc MaxiSorp,
Thermo) were coated with 5 μg/mL recombinant full-length E2
protein in a carbonate-bicarbonate buffer overnight at 4 °C.
All blocking steps were performed using 5% milk protein in PBS for
1 h at 37 °C, and plates were washed with 0.05% Tween-20 in PBS
(PBST) before use. The plates were then incubated with serially diluted
serum samples obtained from mice immunized with CHIKV E2-FL, E2-ΔC,
and E2-ΔNC proteins along with commercially available monoclonal
antibody (18H01) purchased from The Native Antigen (Oxford, United
Kingdom) as a positive control. Serum samples were incubated for 1
h at 37 °C, following which the plates were washed, and CHIKV
E2-specific antibody levels were detected with the HRP-conjugated
goat antimouse IgG secondary antibody (Southern Biotechnology) (1:5000
dilution) for 1 h at 37 °C. Plates were subsequently developed
with 0.4 mg/mL o-phenylenediamine dihydrochloride (SRL, India) dissolved
in citrate-phosphate buffer (pH 5.0). The reaction was stopped with
2 N H2SO4, and optical density at 490 nm was
measured using a microplate reader (Tecan, USA). Titers were defined
as the highest dilution of serum that gave an optical density at least
twice the mean background reading (wells with all reagents except
sera).
E2-specific antibody titers, 96-well ELISA plates (Nunc MaxiSorp,
Thermo) were coated with 5 μg/mL recombinant full-length E2
protein in a carbonate-bicarbonate buffer overnight at 4 °C.
All blocking steps were performed using 5% milk protein in PBS for
1 h at 37 °C, and plates were washed with 0.05% Tween-20 in PBS
(PBST) before use. The plates were then incubated with serially diluted
serum samples obtained from mice immunized with CHIKV E2-FL, E2-ΔC,
and E2-ΔNC proteins along with commercially available monoclonal
antibody (18H01) purchased from The Native Antigen (Oxford, United
Kingdom) as a positive control. Serum samples were incubated for 1
h at 37 °C, following which the plates were washed, and CHIKV
E2-specific antibody levels were detected with the HRP-conjugated
goat antimouse IgG secondary antibody (Southern Biotechnology) (1:5000
dilution) for 1 h at 37 °C. Plates were subsequently developed
with 0.4 mg/mL o-phenylenediamine dihydrochloride (SRL, India) dissolved
in citrate-phosphate buffer (pH 5.0). The reaction was stopped with
2 N H2SO4, and optical density at 490 nm was
measured using a microplate reader (Tecan, USA). Titers were defined
as the highest dilution of serum that gave an optical density at least
twice the mean background reading (wells with all reagents except
sera).
4
Quantifying Sap2-Specific Antibody Titers
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For determining Sap2-specific antibody titers, 96-well ELISA plates (Nunc MaxiSorp, Thermo Scientific, Waltham, MA, USA) were coated with 5 μg/mL of the recombinant Sap2-pp protein in carbonate-bicarbonate buffer overnight at 4 °C. All blocking steps were performed using 5% milk protein in PBS for 1 h at 37 °C and plates were washed with 0.05% Tween 20 in PBS (PBST) before use. The plates were then incubated with serially diluted serum samples obtained from mice immunized with rSap2-pp protein or sham-immunized controls. Serum samples were incubated for 1 h at 37 °C, following which the plates were washed, and Sap2-specific antibody levels were detected with HRP-conjugated goat anti-mouse IgG secondary antibody (Southern Biotechnology, Birmingham, AL, USA) (1:5000 dilution) for 1 h at 37 °C. Plates were subsequently developed with 0.4 mg/mL o-phenylenediamine dihydrochloride (SRL, Gurugram, India) dissolved in citrate-phosphate buffer (pH 5.0). The reaction was stopped with 2N H2SO4 and optical density at 490 nm was measured using a microplate reader (Tecan, Chapel Hill, Morrisville, NC, USA). Titers were defined as the highest dilution of serum that gave an optical density at least twice the mean background reading (wells with all reagents except sera).
5
Coronavirus Antigen-Binding ELISA Assay
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For coronavirus antigen-binding assays, 384-well ELISA plates (Costar #3700) were coated with 2 μg/mL antigens in 0.1M sodium bicarbonate overnight at 4°C. Plates were then washed 1X and blocked for 2 h at room temperature with SuperBlock (1X phosphate buffered saline (PBS) containing 4% (w/v) whey protein 15% normal goat serum/0.5% Tween 20/0.05% sodium azide). Mouse serum samples were collected at baseline before prime, two weeks post prime, four weeks post prime, two weeks post boost, and two weeks post the second boost. Mouse serum samples were added at 1:30 dilution in SuperBlock and diluted 3-fold through 12 dilution spots to generate binding curves. Diluted serum samples were bound to coated plates in SuperBlock for 1h at room temperature. Plates were then washed 2X and a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (SouthernBiotech 1030–05) was added in SuperBlock at a 1:16,000 dilution. Secondary antibody was bound for 1h and then washed 4X and detected with 20μL SureBlue Reserve (KPL 53–00-03) for 15 min. Colorimetric reactions were stopped by adding 20μL of 1% HCL stop solution. Plates were read at 450nm and area under the curve (AUC) was calculated from the serially diluted mouse serum samples.
6
SARS-CoV-2 Antibody Forming Cell Assay
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The plates (Millipore) were coated with antigens (5 μg/mL SARS-CoV-2 Spike protein, or SARS-CoV-2 RBD protein) overnight at 4°C. Then the plates were washed with PBS and blocked using RPMI 1640 medium supplemented with 10% FBS, penicillin/streptomycin, glutamine and 50 μM β-mercaptoethanol for 1h. Bone marrow cells were first treated with ACK lysis buffer to deplete red blood cells. Cells were then plated at 0.25 to 1 million cells per well in RPMI 1640 medium supplemented with 10% FBS, penicillin/streptomycin, glutamine and 50 μM β-mercaptoethanol. After overnight incubation at 37 °C, antigen-specific antibody forming cells were detected by HRP-conjugated goat anti-mouse IgG secondary antibody (1030-05; SouthernBiotech) and IgG2c secondary antibody (1077-05; SouthernBiotech) followed by incubation with 3-amino-9-ethylcarbazole substrate (AEC Substrate Set; Cat: 551951; BD Biosciences). ELISPOT plates were counted manually with magnification glass and normalized as the number of antibody-secreting cells per 1 million bone marrow cells.
7
SARS-CoV-1 and SARS-CoV-2 Antibody Isolation
8
Indirect Binding ELISA Protocol for mAb Characterization
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Indirect binding ELISAs were conducted in 384 well ELISA plates (Costar #3700) coated with 2μg/ml antigen in 0.1M sodium bicarbonate overnight at 4°C, washed and blocked with assay diluent (1XPBS containing 4% (w/v) whey protein/ 15% Normal Goat Serum/ 0.5% Tween-20/ 0.05% Sodium Azide). mAbs were incubated for 60 minutes in three-fold serial dilutions beginning at 100μg/ml followed by washing with PBS/0.1% Tween-20. HRP conjugated goat anti-mouse IgG secondary antibody (SouthernBiotech 1030–05) was diluted to 1:10,000 in assay diluent without azide, incubated at for 1 hour at room temperature, washed and detected with 20μl SureBlue Reserve (KPL 53-00-03) for 15 minutes. Reactions were stopped via the addition of 20μl HCL stop solution. Plates were read at 450nm. Area under the curve (AUC) measurements were determined from binding of serial dilutions.
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