The assay was performed as described[13] . Briefly, polystyrene Micro-ELISA plates (Nunc, Copenhagen, Denmark) were coated overnight with 100 μL of Leishmania donovani soluble antigen at a concentration of 10 μg/mL, diluted in bicarbonate buffer (pH 9.6). Nonspecific binding sites were blocked with 3% bovine serum albumin (BSA) in PBS/0.05% Tween 20 buffer (washing buffer) for 1 hour at 37 °C. The plate was washed 6 times with washing buffer before the addition of 100 μL of the serum samples and incubation for 2 hours at 37 °C. The plate was washed 6 times as above and 100 μL of 1:4000 horse radish peroxidase-conjugated sheep anti-mouse IgG (Amersham) was used as detecting antibody. Tetramethlebenzidine (TMB) microwell peroxidase substrate was added to the wells and the plate was incubated in the dark for 20 minutes before the optical densities were read at 630 nm in a micro-plate reader (Dynatech Laboratories). All sera were tested at a dilution of 1:10, which had been previously determined as the optical dilution for antibody detection by titration.
Horseradish peroxidase conjugated sheep anti mouse igg
Manufactured by Cytiva
Sourced in United Kingdom
Horseradish peroxidase-conjugated sheep anti-mouse IgG is a secondary antibody conjugate used in immunoassay and immunochemistry applications. It consists of sheep-derived antibodies that bind to mouse immunoglobulin G (IgG), coupled with the enzyme horseradish peroxidase.
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3 protocols using horseradish peroxidase conjugated sheep anti mouse igg
1
Leishmania donovani ELISA Antibody Assay
2
Measuring Antibody Response to MUC1 Peptide in Mice
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Sera was collected from C57BL/6 and HLA-A2 transgenic mice 14 or 40 days after final immunisation via orbital bleed. Red blood cells pelleted by centrifugation at 13,000 rpm for 10 min and serum was aspirated and stored at −20 °C until use.
The MUC1 VNTR peptide Cp13-32 (C-PAHGVTSAPDTRPAPGSTAP) was coated onto PVC microtiter plates at 10 µg/mL in 0.2M NaHCO3 buffer, pH 9.6, overnight at 4 °C. After washing (0.05%Tween 20/PBS), non-specific binding was blocked with 2% BSA/PBS for 1 h at RT. Serial dilutions of sera were added (in 2% BSA/PBS) and incubated for a further 2 h at RT. Plates were washed and bound antibody detected using horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham, UK). To determine Ig subclasses plates were washed and bound antibody detected using biotin conjugated sheep anti-mouse IgG1 or IgG2a (BD Pharmingen). Plates were washed as described above and streptavidin HRP added for 1 h at RT. Responses were detected with TMB substrate solution and stopped with 1M HCl. Absorbance was read at 450 nm. End-titre was defined as the last value in the titration to remain above the corresponding control value, where the control was calculated as the mean OD values + 2 SD of naive mouse serum samples (4 mice) at each titration point.
The MUC1 VNTR peptide Cp13-32 (C-PAHGVTSAPDTRPAPGSTAP) was coated onto PVC microtiter plates at 10 µg/mL in 0.2M NaHCO3 buffer, pH 9.6, overnight at 4 °C. After washing (0.05%Tween 20/PBS), non-specific binding was blocked with 2% BSA/PBS for 1 h at RT. Serial dilutions of sera were added (in 2% BSA/PBS) and incubated for a further 2 h at RT. Plates were washed and bound antibody detected using horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham, UK). To determine Ig subclasses plates were washed and bound antibody detected using biotin conjugated sheep anti-mouse IgG1 or IgG2a (BD Pharmingen). Plates were washed as described above and streptavidin HRP added for 1 h at RT. Responses were detected with TMB substrate solution and stopped with 1M HCl. Absorbance was read at 450 nm. End-titre was defined as the last value in the titration to remain above the corresponding control value, where the control was calculated as the mean OD values + 2 SD of naive mouse serum samples (4 mice) at each titration point.
3
Protein Quantification by Immunoblotting
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Total cellular levels of protein were determined by immunoblotting as previously described (Hislop et al., 2004) (link). Briefly, cells were plated into 12-well culture plates and treated as described in figure legends before washing in ice-cold PBS and lysing in extraction buffer (0.5% Triton X-100, 150 mM NaCl, 25 mM KCl, 25 mM Tris pH 7.4, 1 mM EDTA) supplemented with a standard protease inhibitor cocktail (Roche).
Lysates were clarified by centrifugation and resolved by SDS-PAGE using an 8% acrylamide gel and transferred to nitrocellulose and detected using horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Biosciences) or donkey anti-rabbit IgG (Sigma) and enhanced chemiluminesence (ECL) prime detection reagent (Amersham). Band intensities of unsaturated immunoblots were analysed using Peqlab software and quantified by densitometry on ImageJ.
Lysates were clarified by centrifugation and resolved by SDS-PAGE using an 8% acrylamide gel and transferred to nitrocellulose and detected using horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Biosciences) or donkey anti-rabbit IgG (Sigma) and enhanced chemiluminesence (ECL) prime detection reagent (Amersham). Band intensities of unsaturated immunoblots were analysed using Peqlab software and quantified by densitometry on ImageJ.
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