Posters

Traditionally, viruses are purified by time consuming methods such as CsCl density gradient centrifugation or similar. These methods are often inefficient and limited to small scale. In recent years different methods for virus purification, based on ion exchange, gel filtration and affinity chromatography have became popular. Recently, CIM® disk monolithic columns were used for successful concentration of two plant viruses (1) and for improved detection of two human viruses (2). Cucumber mosaic virus (CMV) and Tomato mosaic virus (ToMV) were concentrated and subsequently detected from extremely diluted samples in which they were initially undetectable. Successful concentrations of both viruses encourage us to explore the possibilities of CIM® supports for virus purification. As a model virus ToMV was selected. ToMV is a rod shaped plant virus with a typical size of 300 x 18 nm and isoelectric point at pH 4.6.

The only four drugs approved for the clinical treatment of Alzheirner’s Disease (tacrine. rivastigmine, donepezil and galantamine) are acetylcholinesterase inhibitors which act by maintaining high levels of acetylcholine at the muscarinic and nicotinic receptors in the central nervous system. Human acetylcholinesterase (HuAChE) represents a widely studied target enzyme and it is still object of research for the development of new drugs as enzyme inhibitors.

In a previous paper we reported the immobilisation of AChE on a silica based chromatographic column (50 x 4.6 mm 1.0.) The yeld of immobilization and the stability of the AChE-IMER were considered satisfactory, but some problems arose. The length of the IMER and the large amount of enzyme covalently bound to the chromatographic support resulted in catalysis product long elution times and some inhibitors aspecific matrix absorption with delayed enzyme activity recovery. In order to avoid these complications and considering the high rate of AChE enzymatic reaction. we decided to reduce the dimension of the solid support for immobilization, hence the amount of immobilized enzyme, by selecting a monolithic matrix disk (12 x 3 mm I.D.).

CIM® (Convective Interaction Media) monolithic supports (Biaseparations. Lubiana) represent a novel generation of stationary phases used for liquid chromatography, bioconversions, and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, CIM® supports are cast as continuous homogeneous phases and provide high rates of mass transfer at lower back pressure.

In the present work a CIMK disk with immobilised human recombinant acetylcholinesterase (HuAChE-ClM® Disk) was developed. The activity of immohilised enzyme, the long term stability and reproducibility were tested. HuAChE-CIM® disk was applied as an immobilised enzyme micro-reactor (micro-IMER) in on-line HPLC system for inhibitory potency determination of known AChE inhibitors.