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  • br Acknowledgements br Introduction Autoantibodies directed

    2024-11-08


    Acknowledgements
    Introduction Autoantibodies directed at neurotransmitter receptors and ion channels are pathogenic and/or biomarkers in several autoimmune neurological diseases [1]. For example, autoantibodies directed against voltage-gated potassium channels, the P/Q-type (α1A) voltage-gated calcium channel and the α3 ganglionic nicotinic Cryptochlorogenic acid receptor (AChR) are found in limbic encephalitis, Lambert-Eaton Myasthenic Syndrome (LEMS) and autoimmune autonomic neuropathy, respectively [2], [3], [4]. Sensitive detection of autoantibodies to these neurological receptors is critical to understanding disease pathogenesis, diagnosis and treatment. Detection of these autoantibodies using recombinant proteins in ELISAs is usually not useful because these tests miss important conformational epitopes on these multi-subunit receptors. The most widely used quantitative immunoassay involves using immunoprecipitation of native receptors labeled with radioactive ligands [5]. Alternatively, one can test patient serum by immunohistochemistry with cell lines expressing a given receptor [6], [7], [8]. In Myasthenia Gravis (MG), approximately 85% of patients produce pathogenic autoantibodies which bind and cross link muscle endplate derived nicotinic acetylcholine receptor (AChR). These autoantibodies induce AChR receptor internalization and degradation [2], [9], [10], [11] and/or can activate complement at the cell membrane resulting in cell lysis and destruction of the muscle endplate [12], [13]. Defining the exact targets of autoantibodies in MG has been complicated by the fact that the AChR is composed of five homologous glycoprotein subunits arranged around a central ion pore in the stoichiometry α2βεδ (adult) or α2βγδ (fetal). The four subunits composing the AChR share the same topological structure and all have an N-terminal extracellular domain (∼200 amino acids), four transmembrane segments (∼20 amino acids each) and a short extracellular tail (∼10 amino acids). While the transmembrane segments mediate subunit assembly, the large extracellular domains of the α1, δ and ε (γ in the fetus) subunits contribute to the formation of the two acetylcholine binding sites within the receptor [14]. Lindstrom’s group generated a library of monoclonal antibodies derived from animals immunized against the entire AChR or against chain-specific peptides and used these monoclonal antibodies and radioiodinated AChR to map antigenic determinates on the receptor [15]. One of the most immunodominant regions on the receptor was a 10 amino acid peptide sequence within the extracellular domain of the AChR-α1 subunit, designated the Main Immunogenic Region (MIR) [15], [16]. Competition experiments with these defined monoclonal antibodies also demonstrated that a significant number of MG patients had autoantibodies against the MIR [17]. Additionally, passive transfer of antibodies directed at the MIR also induces experimental autoimmune Myasthenia Gravis in rats [18]. Although the AChR-α1 subunit appears to be the major target of autoantibodies in MG, it is possible that other chains alone or in combination with the α1 chain are required for detecting some MG patient autoantibodies. Clinical detection of MG usually involves immunoprecipitation of native receptor labeled with radioactive α-bungarotoxin (α-BTX), a snake venom-derived toxin that binds the AChR outside the ligand binding domain with high affinity (KD=2.6×10−10M) [19]. In these immunoassays, native pentameric AChR from human sources is first partially purified, radioactively labeled with 125I-α-bungarotoxin and then used in radioimmunoprecipitation assay (RIA) to measure patient autoantibodies [20]. Immunohistochemical analysis using cell lines expressing the various AChR receptor subunits has been successfully used to detect patient autoantibodies [8]. Immunoassays using peptides or recombinant AChR proteins have also been tried [21], [22]. Recombinant, bacterially-expressed AChR proteins react poorly with MG patient sera, suggesting that most MG patient autoantibodies recognize conformational rather than linear epitopes within the extracellular domain of the AChR-α1 subunit. Other assay formats employing non-radioactive labeling of the receptor require cumbersome purification methods [23], [24].