A radioisotope label-free alpha-bungarotoxin-binding assay using BIAcore sensor chip technology for real-time analysis

Abstract

alpha-Bungarotoxin (alpha-bgtx)-binding proteins, including certain nicotinic acetylcholine receptors and acetylcholine-binding proteins (AChBPs), are frequently characterized with radioisotope-labeled alpha-bgtx-binding assays. Such assays, however, preclude investigations of binding interactions in real time and are hampered by the inconveniences associated with radioisotope-labeled reagents. We used surface plasmon resonance-based technology (BIAcore) to investigate the binding of recombinant AChBP to CM-5 sensor chip surfaces with directly immobilized alpha-bgtx. We validated our BIAcore results by comparing the same biological samples using the traditional (125)I-labeled alpha-bgtx-binding assay. An alpha-bgtx sensor chip, as described here, enables detailed, real-time, radioisotope-free interaction studies that can greatly facilitate the characterization of novel alpha-bgtx-binding proteins and complexes.

Fig. 1. Concentration-dependence of signal responses due to AChBP binding to the α-bgtx sensor chip

Purified AChBP was injected (from point a to point b) at concentrations ranging from 0 to 100 μg/ml. Point c indicates the starting point for the regeneration of the chip surface with 1 M carbachol.

Fig. 2. Detecting AChBP expression in Pichia pastoris culture media

This bar graph compares the assay data obtained with the BIAcore α-bgtx sensor chip (white bars) with data from an [¹²⁵I]-α-bgtx-binding assay (gray bars). BIAcore and [¹²⁵I]- α-bgtx-binding assay data were normalized to the value of the sample with the greatest response at point c (see Fig. 1) and the sample with the maximum radioactive counts, respectively.