Luciferase Immunoprecipitation Systems LIPS which utilizes r

Luciferase Immunoprecipitation Systems (LIPS), which utilizes recombinant antigens fused to the enzyme reporter Renilla luciferase (Ruc) to detect patient antibodies, provides a unique platform to investigate dcp cas directed against a variety of antigenic targets [25]. Previously, LIPS has been used to efficiently evaluate autoantibody responses in several autoimmune diseases mainly targeting soluble human autoantigens [25]. Here we investigated whether LIPS could be used to evaluate autoantibodies associated with the single AChR-α1 chain in MG. Using a series of deletion mutants, the antigenicity of the AChR-α1 subunit was systematically studied with an anti-AChR-α1 monoclonal antibody, control and MG patient serum samples. From these studies, statistically significant levels of autoantibodies against the AChR-α1 subunit were detected in 32% of MG patients. This approach, employing single subunits of the AChR to detect patient autoantibodies may provide a potentially useful method of evaluating patient autoantibodies to other neurotransmitter receptors and ion channels in other autoimmune neurological diseases.
Materials and methods
Results
Discussion Antibodies directed at neurotransmitter receptors and ion channels have been recognized in several autoimmune channelopathies, including LEMS, paraneoplastic cerebellar ataxia, limbic encephalitis and MG [1]. To date, immunoprecipitation assays using native receptor or ion channels, radiolabeled with specific ligands, remain the primary sensitive method of detecting patient autoantibodies in the clinical setting. Here we have used the LIPS assay, employing single chain receptor antigen fusions, to study autoantibodies in MG. From screening a panel of recombinant Ruc-AChR-α1 fusions, the different AChR fusion extracts showed a large range of immunoreactivity (0–43%), despite the fact that they all contained the immunogenic extracellular domain. The findings that some AChR-Ruc fusions showed high levels of MG patient immunoreactivity while other did not, highlight the variable and unpredictable nature of recapitulating the needed conformational epitopes associated with this receptor chain. This reductionistic approach provides a route to isolating important factors that shape the immune responses in MG and possibly the clinical course of disease. Comparison of the single AChR-α1 chain LIPS assay with the established RIA employing 125I-α-BTX-labeled native receptor revealed that the RIA showed 63% sensitivity, while LIPS showed 32% sensitivity. One possible reason for the markedly higher sensitivity of the RIA compared to LIPS is that RIA uses the native receptor containing three additional subunits (β, δ and γ/ε subunits) besides the alpha chain. It is likely that these other AChR subunits are required for optimal folding and detection of conformational autoantibodies that are missed by LIPS. Consistent with this idea, a chimera in which the MIR of the AChR-α1 subunit was incorporated into the AChR-α7 subunit and which was immunoprecipitated by monoclonal antibodies directed at the MIR, failed to directly bind autoantibodies in MG patient sera, suggesting the importance of specific contributions from the other subunits of the muscle derived AChR in the detection of autoantibodies in some patients [30]. Another possibility accounting for the lower sensitivity of the LIPS assay compared to the α-BTX RIA is that the AChR-α1 protein fusions expressed in the LIPS assay may still not fold optimally. This may be because the AChR-α1 chain is normally a transmembrane protein and its artificial expression as a fusion with Ruc may disrupt its native conformation. Along these lines, autoantibody immunoreactivity toward the NMDA receptor was originally thought to involve multiple receptor subunits, but was later shown to be directed toward a single subunit [6], [7], [31]. These studies of the AChR and NMDAR highlight the complexity in identifying and recapitulating conformational epitopes directed at neurotransmitter receptors to detect patient autoantibodies and give further incentive to develop a method to systematically evaluate epitopes to specific receptor subunits.