Biotinylated O-linked saccharides (1–100 ng as total CS disaccharides) were immobilized on a 96-well BD BioCoat streptavidin assay plate (BD Biosciences; Deepa et al., 2002 (link)). After blocking with 2% BSA in PBS, Cat316 antibody (diluted 20,000-fold with 2% BSA in PBS) was added to each well and incubated at 4°C overnight. After washing with PBS, peroxidase-conjugated anti-mouse IgG+M (1/10,000 in 2% BSA/PBS) was added to the wells and incubated for 2 h at room temperature. After washing, the color was developed using a ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) substrate kit (Vector Laboratories) and the optical density was measured at 415 nm using a microplate reader (Bio-Rad model 550).
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts)
Manufactured by Vector Laboratories
ABTS is a colorimetric assay reagent used to measure antioxidant activity. It produces a stable blue-green colored radical that can be quantified spectrophotometrically.
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Lab products found in correlation
2 protocols using 2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts)
1
Quantification of Chondroitin Sulfate Binding
2
Phage-antibody binding ELISA
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Purified MS brain 95–2 IgG in 0.1 M carbonate buffer (50 μl, 200 μg/ml) was coated onto wells of ELISA plates overnight at 4 °C. The wells were then blocked with 3% BSA for 2 h followed by incubation with purified phage 3–2 and 3–3 (5 × 109/well) for 1 h. After washing with 0.05% Tween-20/Tris buffered saline (TBST), the wells were incubated with a 1:500 dilution of mouse anti-M13 IgG-HRP (New England BioLabs) antibody for 1 h, followed by incubation with peroxidase substrate ABTS (Vector Laboratories, Burlingame, CA) for 20 min. The optical absorbance was measured at 415 nm with a Microplate Manager (BioRad, Hercules, CA). All samples were tested in duplicate and the ELISA was repeated at least one additional time.
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