For detecting NP-specific antibodies in sera, wells were coated with 1 μg/mL NP23-BSA or NP2-BSA in coating buffer (Bicarbonate-carbonate buffer, pH 9.6) overnight. Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37°C for 1 h, washed twice, and incubated with indicated sera samples at 37°C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37°C for 1 h after washing with a buffer for four times. The reaction was further developed with tetramethylbenzidine (TMB), then stopped by 2N H2SO4, and read at 450 nm. Sera from influenza-challenged mice were assayed using total viral lysate prepared from stocks of strain Influenza A/PR8/34.
Blocking protein
Manufactured by Bio-Rad
5% blocking protein is a laboratory reagent used to minimize non-specific binding in various immunoassay and protein detection techniques. It provides a simple and effective way to block non-specific binding sites, helping to improve signal-to-noise ratios and enhance the specificity of your assays.
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2 protocols using blocking protein
1
Detecting NP-specific Antibodies in Sera
2
Detecting NP-specific Antibodies in Sera
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For detecting NP-specific antibodies in sera, wells were coated with 1 μg/mL NP23-BSA or NP2-BSA in coating buffer (Bicarbonate-carbonate buffer, pH 9.6) overnight. Plates were washed twice with washing buffer (0.05% Tween 20 in PBS), blocked with 5% blocking protein (Bio-Rad) at 37°C for 1 h, washed twice, and incubated with indicated sera samples at 37°C for 1.5 h. Horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, and anti-mouse IgG3 (all from SouthernBiotech), or anti-mouse IgM (Bethyl laboratories) were developed at 37°C for 1 h after washing with a buffer for four times. The reaction was further developed with tetramethylbenzidine (TMB), then stopped by 2N H2SO4, and read at 450 nm. Sera from influenza-challenged mice were assayed using total viral lysate prepared from stocks of strain Influenza A/PR8/34.
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