Lundonia Biotech

TechNotes No. 1-1999

Binding and inhibition of a Concanavalin A — horseradish peroxidase conjugate to a–D–mannopyranoside 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 and inhibition of a a–D–mannopyranoside recognizing lectin¹, Concanavalin A, to a–D-mannopyranoside GlycoWell™ plates.

Experimental

Addition of inhibitor.Add to three wells of each GlycoWell™ plate 50µL of inhibitor solution and to three wells of each GlycoWell™ plate 50µL of TWEEN/PBS.
Lectin incubation. Add 50µL 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 100µL substrate solution to each well. Read optical density at 490 nm in a microwell plate reader.

Results
The results demonstrate that a a–D-mannopyranoside GlycoWell™ plate efficiently binds a mannose–recognizing plant lectin, Concanavalin A (Figure 1). Thus, GlycoWell™ plates are suitable for detecting carbohydrate binding properties of proteins. Furthermore, a soluble a–D-mannopyranoside inhibits the binding of the Concanavalin A–horseradish peroxidase conjugate to the a–D–mannopyranoside GlycoWell plate, demonstrating the suitability of using GlycoWell™ plates in competitive inhibition experiments.

Materials

GlycoWell™ plates SW-00-001 (blank) and SW-01-009 (a–D-mannopyranoside).
PBS buffer pH 6.8, 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.
Inhibitor solution: 2 mM of 2–(2–carboxyethylthio)–ethyl a–D–mannopyranoside in TWEEN/PBS.
Lectin stock: 1.0 mg Concanavalin A–horseradish peroxidase conjugate (SIGMA L6397), 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.

Figure 1. A Concanavalin A-horseradish peroxidase conjugate binds to a–D–mannopyranoside GlycoWell™ plates (SW-01-009), but not to a GlycoWell™ plate devoid of saccharide (SW-00-001). A soluble a–D-mannopyranoside inhibits the binding of the Concanavalin A–horseradish peroxidase conjugate to the a–D-mannopyranoside GlycoWell™ plate.

Reference
1. Reeke, G. N. et al. Ann. N.Y. Acad. Sci.,1974, 234, 369.