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AIM: Antibodies against voltage-gated calcium channels (VGCC) cause the Lambert-Eaton myasthenic syndrome and are often indicative for the presence of a lung carcinoma. There is an urgent need to develop a non-radioactive method for detecting these antibodies. We propose to create a rapid and efficient method for the diagnosis of the Lambert-Eaton myasthenic syndrome using fluorescently-tagged VGCC.
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Situation
The need for a new assay. Despite our expertise and the success of the present diagnostic assay its future is uncertain. The standard diagnostic test for LEMS has to be carried out in a laboratory designated for the use and safe disposal of radioactive materials, registered by the Environmental Agency. There is a general movement to eliminate techniques involving radioactive materials because of safety and regulatory concerns. Recently we have learnt that GE Healthcare, the only European manufacturer of 125I-CmTx, is to stop supplying radioiodinated compounds, which will include radiolabelled conotoxins from December 13th 2007. We are now entirely dependent on one supplier, the American company PerkinElmer Inc., based in Massachusetts, who could similarly stop production, without warning, at any time. A novel technique for detecting anti-voltage-gated calcium channel serum antibodies. We have recently developed a new method for labelling complex proteins such as ion channels using fluorescent tags. ‘Enhanced green fluorescent protein’, EGFP, can frequently be incorporated into proteins with little effect on their conformation. This provides a mechanism for ‘tagging’ a protein without the need for neurotoxins or radioactivity. When solubilised these fluorescently tagged proteins can be used in conventional immunoprecipitation experiments to detect serum antibodies. This technique has been successfully piloted in our department to detect the binding of sera from myasthenia gravis patients to fluorescently tagged muscle AChR (see figure). The sensitivity of this fluorescence ‘read-out’ is similar to results obtained using radioactive -bungarotoxin, which is used in the radioactive assay for anti-AChR antibodies.