High binding ELISA plates (Thermo Scientific, catalog no 44-2404-21) were coated at 0.5μg/mL with goat anti-mouse IgG2b (Southern Biotech, catalog no 1090–05) overnight at 4°C. Next day, plates were washed 6x with PBST (PBS + 0.05%Tween-20) and blocked with 1% BSA in PBST for 30 minutes at RT. Anti-p27 2F12 (NIH AIDS Reagent Program) antibody at 0.5 μg/mL was added to ELISA plates and incubated for 1 hr at 37°C. Plates were again washed 6x with PBST. Supernatant from ADCVI assays which were placed in the 4°C the night before, were treated with TritonX (Sigma) to a make a 0.5% Triton X solution to inactivate virus particles and release Gag. Gag supernatant was diluted 3-fold and added to ELISA plates and allowed to incubate for 1½ hr at 37°C. Plates were washed and biotinylated anti-SIV IgG diluted 1:1000 and added to each well. Plates were again incubated for 1 hr at 37°C. After 6x wash, neutralite-avidin peroxidase (N-HRP) (Southern Biotech) was diluted 1:4000 and added to each plate for 30 minutes at RT in the dark. After incubation, bound IgG was detected using tetramethylbenzidine substrate (KPL, Gaithersburg, MD). The reaction was stopped by adding 100 μl 2N H2SO4. The readings were recorded at 450nm.
Neutralite avidin peroxidase n hrp
Manufactured by Southern Biotech
Neutralite-avidin peroxidase (N-HRP) is a laboratory reagent used in various biotechnological and immunological applications. It consists of avidin, a protein with a high affinity for biotin, conjugated to the enzyme horseradish peroxidase (HRP). This conjugate is designed to facilitate the detection and quantification of biotinylated molecules in experimental procedures.
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2 protocols using neutralite avidin peroxidase n hrp
1
Quantification of Anti-SIV Antibodies
2
Quantification of Anti-SIV Antibodies
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High binding ELISA plates (Thermo Scientific, catalog no 44-2404-21) were coated at 0.5μg/mL with goat anti-mouse IgG2b (Southern Biotech, catalog no 1090–05) overnight at 4°C. Next day, plates were washed 6x with PBST (PBS + 0.05%Tween-20) and blocked with 1% BSA in PBST for 30 minutes at RT. Anti-p27 2F12 (NIH AIDS Reagent Program) antibody at 0.5 μg/mL was added to ELISA plates and incubated for 1 hr at 37°C. Plates were again washed 6x with PBST. Supernatant from ADCVI assays which were placed in the 4°C the night before, were treated with TritonX (Sigma) to a make a 0.5% Triton X solution to inactivate virus particles and release Gag. Gag supernatant was diluted 3-fold and added to ELISA plates and allowed to incubate for 1½ hr at 37°C. Plates were washed and biotinylated anti-SIV IgG diluted 1:1000 and added to each well. Plates were again incubated for 1 hr at 37°C. After 6x wash, neutralite-avidin peroxidase (N-HRP) (Southern Biotech) was diluted 1:4000 and added to each plate for 30 minutes at RT in the dark. After incubation, bound IgG was detected using tetramethylbenzidine substrate (KPL, Gaithersburg, MD). The reaction was stopped by adding 100 μl 2N H2SO4. The readings were recorded at 450nm.
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