The lectin reactivity of the TF-specific IgG was measured by ELISA in a similar way, except that the binding of neuraminic acid- (sialic acid-) specific Sambucus nigra agglutinin (SNA) and mannose-specific concanavalin A (ConA) to the absorbed serum anti-TF Abs (all isotypes) or anti-TF IgG from tIgG samples was measured as described elsewhere [19 (link), 21 (link)]. Biotinylated SNA (Vector Laboratories Inc., USA) in 10 mmol/L HEPES, 0.15 mol/L NaCl, and 0.1 mmol/L CaCl2, pH 7.5, and biotinylated ConA (Sigma, USA) in the ConA binding buffer (0.05 mol/L Tris-HCl buffer, pH 7.2, containing 0.2 mol/L NaCl and 3 mmol/L CaCl2, MgCl2, and MnCl2) were both applied at a concentration of 5 μg/mL each, for 1.5 h at 25°C. The bound lectins were detected with a streptavidin-alkaline phosphatase conjugate (Dako, Denmark) and p-nitrophenylphosphate (Sigma, USA). The optical density value of control wells (no sample) was subtracted from that of Ab-coated wells to determine the lectin binding. Each sample was analyzed in duplicate.
Streptavidin alkaline phosphatase conjugate
Manufactured by Agilent Technologies
Sourced in Denmark, United States
Streptavidin-alkaline phosphatase conjugate is a detection reagent that combines the high affinity of streptavidin for biotin with the enzymatic activity of alkaline phosphatase. This conjugate is commonly used in various bioanalytical techniques, such as ELISA and Western blotting, to amplify and detect the presence of biotinylated targets.
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4 protocols using streptavidin alkaline phosphatase conjugate
1
Lectin Reactivity of Anti-TF IgG
2
TF-Glycotope Antibody Lectin-Reactivity Assay
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The SNA lectin-reactivity of TF-glycotope specific antibodies was measured in a similar way.
The plates (Maxisorp, NUNC, Denmark) were coated with synthetic TF polyacrylamide conjugate (10 mol% of carbohydrate; Lectinity, Russia) in carbonate buffer, pH 9.6, 5 μg per well. After overnight incubation at +4°C, triple washing and blocking with Superblock solution (Pierce, USA) for 30 min at 25°C, the serum samples (diluted 1 : 25 in PBS-0.05% Tween) were applied for 1.5 hr at 25°C. After subsequent washing with PBS-Tw, the biotinylated SNA (Vector Laboratories Inc., USA) in 10 mmol/L Hepes, 0.15 mol/L NaCl, 0.1 mmol/L CaCl2, and pH 7.5 was applied at a concentration of 5 μg/mL for 1.5 hr at 25°C. The bound lectin was detected with a streptavidin-alkaline phosphatase conjugate (Dako, Denmark) and p-nitrophenylphosphate (Sigma, USA). The optical density value (OD) of control wells (no sample) was subtracted from the Ab coated wells. Each sample was analysed in duplicate.
The plates (Maxisorp, NUNC, Denmark) were coated with synthetic TF polyacrylamide conjugate (10 mol% of carbohydrate; Lectinity, Russia) in carbonate buffer, pH 9.6, 5 μg per well. After overnight incubation at +4°C, triple washing and blocking with Superblock solution (Pierce, USA) for 30 min at 25°C, the serum samples (diluted 1 : 25 in PBS-0.05% Tween) were applied for 1.5 hr at 25°C. After subsequent washing with PBS-Tw, the biotinylated SNA (Vector Laboratories Inc., USA) in 10 mmol/L Hepes, 0.15 mol/L NaCl, 0.1 mmol/L CaCl2, and pH 7.5 was applied at a concentration of 5 μg/mL for 1.5 hr at 25°C. The bound lectin was detected with a streptavidin-alkaline phosphatase conjugate (Dako, Denmark) and p-nitrophenylphosphate (Sigma, USA). The optical density value (OD) of control wells (no sample) was subtracted from the Ab coated wells. Each sample was analysed in duplicate.
3
Lectin Reactivity of TF Glycotope Antibodies
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The lectin reactivity of TF glycotope specific antibodies was measured in a similar way, except that the binding of the neuraminic acid (sialic acid) specific Sambucus nigra agglutinin (SNA) to the absorbed anti-TF antibodies was determined as described by Kodar et al. [23 (link)]. The biotinylated SNA (Vector Laboratories Inc., USA) in 10 mmol/L Hepes, 0.15 mol/L NaCl, 0.1 mmol/L CaCl2, and pH 7.5 was applied at a concentration of 5 μg/mL for 1.5 h at 25°C. The bound lectin was detected with a streptavidin-alkaline phosphatase conjugate (Dako, USA) and p-nitrophenylphosphate (Sigma, USA). The optical density value (O.D.) of control wells (no sample) was subtracted from that of Ab-coated wells to determine the lectin binding. Each sample was analysed in duplicate. The value of the SNA binding to all TF-specific Abs and the ratio of SNA binding to TF-specific IgG, IgM, and IgA level (SNA/Ig index) were determined.
4
IgE-Binding Proteins Identification in Insect Allergens
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Affinity-purified Periplaneta americana protein (50 mg) was separated in a 12% SDS-PAGE in a Mini-Protean 3 Cell (Bio-Rad) and electroblotted onto a PVDF membrane (Amersham Biosciences, Bucks, UK) that had been cut into vertical strips. The strips were blocked with 3% BSA in TBS at 25°C for 1 hr and then incubated individually with the sera of allergic patients or controls (diluted 1:5) at 4°C overnight. After rinsing with TBS-T, the strips were placed in a solution of mouse antihuman IgEÀbiotin conjugate (Zymed) diluted 1:1000 and incubated at 25°C for 3 hr, washed with TBS-T, and incubated with streptavidin-alkaline phosphatase conjugate (DakoCytomation, Carpinteria, CA, USA) diluted 1:1000 in diluent at 25°C for 30 min, then washed. Finally, the strips were incubated with nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate substrate in the dark for 10 min. The enzymeÀsubstrate reaction was stopped by rinsing all strips with deionized distilled water.
For the immunoblot inhibition assay, 150 mg crude extract of silkworm, moth or cockroach was blotted onto a nitrocellulose membrane and then reacted with pooled sera (1:5 diluted from patients) that had been preincubated with 10 mg purified recombinant Bom m 9 or moth or cockroach for 1 hr at room temperature.
For the immunoblot inhibition assay, 150 mg crude extract of silkworm, moth or cockroach was blotted onto a nitrocellulose membrane and then reacted with pooled sera (1:5 diluted from patients) that had been preincubated with 10 mg purified recombinant Bom m 9 or moth or cockroach for 1 hr at room temperature.
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