Identification Of A Novel Antibody In Autoimmune Encephalitis

BackgroundAutoimmune diseases in nervous system are caused by autoimmune molecules and immune cells attacking the nervous system,which can occur in the central nervous system,peripheral nervous system,and nerve-muscle junctions.Among of them,autoimmune encephalitis(AE)and central nervous system demyelinating diseases are the most.Target autoantibodies are the critical factors for the understanding the disease,and play an important role in the diagnosis and treatment of patients.Take AE as an example.Autoimmune encephalitis(AE)is one of the intractable and severe neurological diseases,characterized by acute or subacute onset,diverse clinical symptoms,high mortality and disability rate.In the past ten years,it has been discovered that autoantibodies against neuronal cell surface proteins or synaptic proteins mediate immune responses and cause neuronal dysfunction.Early immunotherapy is effective.The detection of specific autoantibodies is required for the definite diagnosis of AE.Nearly 20 types of autoantibodies have been found for AE.The clinical features and early diagnosis and interpretation are still in the early stage,and the total detection rate is less than 20%.Unknown antibodies are also urgently waiting to be discovered.This project further analyzes the samples of patients with suspicious AE,whose screening of known AE antibodies tests were negative.The purpose of the study is to search for new AE antibodies,and explore the clinical diagnostic value of new antibodies through reduction experiments.ObjectivesTo discover new antibodies for AE and clarify its diagnostic value.At present,the detection rate of total antibodies for suspicious AE is 10-20%.Find new antibodies for patients with negative known antibody tests to provide a basis for further clarifying the diagnosis of AE,elucidating the pathogenesis,and seeking more reasonable and effective treatment measures.The samples of patients with suspicious AE whose screening of known AE antibodies tests are negative were used.Methods1.Retrospective research and application for ethical approval2.Criteria for inclusion of cases:1)Time:Inpatients in Nanfang Hospital between 2018-01-01 to 2020-12-312)Clinical symptoms or auxiliary examinations are consistent with suspicious cases of autoimmune encephalitis3)The result of the known antibody test of autoimmune encephalitis by the cellular immunological double fluorescence method is negative4)Use tissue section method to detect immune response5)There are remaining specimens of cerebrospinal fluid and/or serum 3.Use immunoblotting,co-immunoprecipitation and other methods for candidate cases to find the corresponding bands of unknown antibodies.Analyze the protein of the corresponding band by liquid chromatography mass spectrometry to identify the nature of the unknown antibody.4.Reduction verification:1)Use the HER293T cell immunofluorescence method,immunoprecipitation,immunoblotting method,and primary neuron confocal localization to verify the target protein overexpressed.2)New antibody screening for more suspected cases5:Epitope:Detect the reactivity of the patient’s antibody to each subunit of the target protein by CBA.Results1.In a patient suspected of autoimmune encephalitis,autoantibodies against neuron CRMP2(unpublished papers and patents,use code CRMP2,the same below)were identifiedWe included a 38-year-old woman(patient P1)who was admitted to the hospital due to dizziness,blurred vision,unstable walking,and fever for more than 8 days.Known autoantibody tests during hospitalization were all negative,but the patient’s cerebrospinal fluid samples were subjected to immunohistochemical staining of mouse brain tissue sections,which showed that the brain tissue showed extensive and diffuse hyperresponsiveness.We used patients’ cerebrospinal fluid and serum samples to perform immunoprecipitation and mass spectrometry analysis of mouse whole brain proteins,and identified the antigen as CRMP2.2.The reduction experiment verifies that the antibody in the P1 serum/cerebrospinal fluid sample is anti-CRMP2 antibody.We used TBA,CBA,and immunoprecipitation to verify the antibody pairs in P1 patient samples the specificity of CRMP2.3.Among the other 72 suspected AE cases included,2 more patients with CRMP2 antibody were found.The CBA method was used to screen 72 other suspected AE cases,and the same antibody was detected in the other 2 suspected AE patients.4.The CRMP2 antibody mainly recognizes the V3 subunitWe detect the immune response of the patient’s serum antibodies to the three subunits of CRMP2,V1,V2,and V3 by CBA,and the results showed that the main epitope of CRMP2 is located in V3.ConclusionsIn patients with autoimmune encephalitis,autoantibodies against CRMP2 were identified and their diagnostic value was clarified.The symptoms of 3 patients were significantly improved after immunotherapy.Therefore,the detection of CRMP2 autoantibodies in patients with suspected autoimmune encephalitis has important diagnostic and therapeutic guidance significance.