Immunosensor for detection of inhibitory neurotransmitter gamma-aminobutyric acid using quartz crystal microbalance.

A piezoelectric immunosensor for sensing the low molecular weight neurotransmitter gamma-aminobutyric acid (GABA), one of two major inhibitory neurotransmitters in the central nervous system, is described. The sensing interface consists of a dextran layer covalently attached to a self-assembled monolayer of thiolamine compound on the surface of gold electrodes of the crystals. The dextran layer is further modified with GABA molecules to act as the biosensing layer. The affinity binding of monoclonal anti-GABA antibody on the modified piezoelectric crystals is studied in real time without any additional labels. The equilibrium association constant, K(eq) for binding between anti-GABA antibody and GABA molecules is 14.5 microg x mL (-1). The detection limit for anti-GABA is approximately 10 nM. The sensitivity of the sensor at a concentration corresponding to half-maximal response is 13.6 ng/mL x Hz. The functionalized sensor substrate is subsequently used for competitive determination of different concentrations of free GABA (range of 5 microM-50 mM) in PBS-BSA buffer. The detection limit of the immunosensor for sensing GABA with maximum sensitivity is approximately 42 microM.

Inhibition of gamma-aminobutyric acid synthesis by glutamic acid decarboxylase autoantibodies in stiff-man syndrome.

Stiff-man syndrome (SMS) is a rare disorder of the central nervous system thought to result from an impairment of gamma-aminobutyric acid (GABA)ergic neurotransmission. Autoantibodies to the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), present in about 60% of SMS patients, have suggested an autoimmune pathogenesis of SMS. By using serum or cerebrospinal fluid from 25 SMS patients, we assessed the effect of GAD autoantibodies (GAD-A) on GAD enzymatic activity in vitro; 83% of GAD-A-positive SMS sera reduced GABA production in crude rat cerebellar extracts, whereas GAD-A- sera from SMS patients or healthy blood donors did not alter the enzyme activity. Inhibition of GABA synthesis by SMS sera was dose dependent and mediated by the purified IgG fraction of the sera. Human monoclonal GAD65-A and IgG purified from serum of GAD-A-positive patients with insulin-dependent diabetes or autoimmune polyendocrine syndrome did not affect GAD activity, suggesting that a specific epitope recognition of GAD-A mediates inhibition of GAD. The disease-specific detection of GAD-inhibitory antibodies is compatible with their functional involvement in the etiopathology of SMS; the relevance of such antibodies in vivo, however, remains to be determined.