TechNotes No. 4-1999

Binding of a Bandeiraea simplicifolia agglutinin — horseradish peroxidase conjugate to b–Globotrioside GlycoWell™ plates.

Introduction
Binding of lectins to immobilized saccharides and subsequent inhibition of such a binding event is often used for evaluating lectin inhibitors. The use of GlycoWell™ plates facilitates an ELISA assay involving the binding of lectins to the wells. This TechNote describes the binding of a a–D–galactopyranoside recognizing lectin, Bandeiraea simplicifolia agglutinin (bs–I)¹, to b–globotrioside (Gb3) GlycoWell™ plates.

Experimental

1. Lectin incubation. Add 100 µL lectin solution to each well. Leave for 45 min
2. Washing. Empty the wells and wash three times with CovaBuffer. Leave the last wash in the wells for 15 min. Wash with citrate/phosphate buffer once.
3. Substrate reaction. Add 100 µL substrate solution to each well. Read optical density at 490 nm in a microwell plate reader.

Results
The results demonstrate that a b–globotrioside GlycoWell™ plate efficiently binds a a–D–galactopyranoside–recognizing plant lectin, Bandeiraea simplicifolia agglutinin (bs–I) (Figure 1). Thus, GlycoWell™ plates are suitable for detecting carbohydrate binding properties of proteins.

Materials

• GlycoWell™ plates SW-00-001 (blank) and SW-03-002 (b–globotrioside.)
• PBS buffer pH 7.2, 0.15 M Na⁺: 24.0 g NaCl, 0.6 g KCl, 3.46 g Na₂HPO₄·2H₂O, 0.6 g KH₂PO₄, 3 L H₂O.
• TWEEN/PBS: 0.10 mL TWEEN 20, 200 mL PBS.
• CovaBuffer: 116.9 g NaCl, 10.0 g MgSO₄·7H₂O, 0.50 mL TWEEN 20, 1 L PBS.
• Citrate–Phosphate buffer, pH 5.0: 7.30 g citric acid, 11.86 g Na₂HPO₄·2H₂O, 1 L H₂O.
• Lectin stock: 1.0 mg Bandeiraea simplicifolia agglutinin (bs–I)–horseradish peroxidase conjugate (SIGMA L3383), 1.0 mL TWEEN/PBS.
• Lectin solution: 70 µl Lectin stock, 1.75 ml TWEEN/PBS, filtered through a 0.22 micron filter.
• Substrate solution: 6.0 mg o–phenylenediamine, 10.0 mL citrate/phosphate buffer, 5 µL H₂O₂, freshly prepared.
• 1 M H₂SO₄

Figure 1. A Bandeiraea simplicifolia agglutinin (bs–I)lectin–horseradish peroxidase conjugate binds GlycoWell™ plates (SW-03-002), but not to a GlycoWell plate devoid of saccharide (SW-00-001).