Lundonia Biotech

TechNotes No. 3-1999

Binding of plant lectin — horseradish peroxidase conjugate to b–lactoside GlycoWell™ plates.

Introduction
Binding of lectins to immobilized saccherides 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 three b–lactose recognizing lectins, Erythrina cristagalli lectin¹, peanut agglutinin², and Ricinus communis toxin RCA 120³, to b–lactoside GlycoWell™ plates.

Experimental

Lectin incubation.Add 100 µL lectin solution to each well. Leave for 45 min
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.
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 b–lactoside GlycoWell™ plate efficiently binds different b–lactose–recognizing plant lectins, (Figure 3). Thus, GlycoWell™ plates are suitable for detecting carbohydrate binding properties of proteins.

Materials

GlycoWell™ plates SW-00-001 (blank) and SW-02-001 (b–lactoside).
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 of lectin [E. cristagalli–horseradish peroxidase conjugate (SIGMA L9050), peanut agglutinin–horseradish peroxidase conjugate (SIGMA L7759), RCA120–horseradish peroxidase conjugate (SIGMA L2758) in 1.0 mL TWEEN/PBS.
Lectin solution: 70 µL Lectin stock, 1.75 mL TWEEN/PBS, filtered through a 0.22 micron filter. In most cases the concentration of lectin must be optimized to obtain good readings
Substrate solution: 6.0 mg o–phenylenediamine, 10.0 mL citrate/phosphate buffer, 5 µL H₂O₂, freshly prepared.
1 M H₂SO₄

Figure 1: Three different b–lactoside–recognizing lectin–horseradish peroxidase conjugates bind to b–D–lactopyranoside GlycoWell™ plates (SW-02-001), but not to a GlycoWell™ plate devoid of saccharide (SW-00-001).

Reference
1. Iglesias, J.L., Eur. J. Biochem., 1982, 123, 247.
2. Bird, G.W.G., Vox Sang., 1964, 9, 748.
3. Nicolson, G.L. and Blaustein, J. Biochim. Biophys. Acta, 1972, 266, 543.