Antibodies In Autoimmune Diseases Research Articles

Vibrance-mg: Clinical Trial of Nipocalimab in Pediatric Myasthenia Gravis

Objective We describe an open-label study of nipocalimab to determine the effect of nipocalimab in pediatric participants with gMG. Background Nipocalimab is a high affinity, fully human, aglycosylated, effectorless IgG1 anti FcRn monoclonal antibody that targets the neonatal Fc receptor (FcRn) with high affinity, thereby lowering IgG pathogenic antibodies in autoimmune disease. Data from Vivacity-MG, a Phase 2, multicenter, randomized, double-blind, placebo-controlled study of nipocalimab demonstrated safety, tolerability, and efficacy of nipocalimab in adult generalized myasthenia gravis (gMG) (clinicaltrials.gov NCT03772587). Design/Methods This global study will enroll at least 12 participants, aged 2 to <18, with a diagnosis of gMG and an insufficient clinical response to ongoing, stable standard-of-care therapy, as reflected by a Myasthenia Gravis Foundation of America (MGFA) Class of IIa/b through IVa at screening. Participants must have a positive serologic test for either acetylcholine receptor or Muscle Specific Tyrosine Kinase pathogenic autoantibody. The study will consist of a screening period of up to 4 weeks, a 24-week open-label Active Treatment Phase where participants will receive nipocalimab intravenously every two weeks, and a Long-term Extension phase; after last dose, a safety follow-up assessment will be conducted at 8 weeks. The primary outcome is the effect of nipocalimab on total serum IgG, safety and tolerability, and PK in pediatric participants with gMG. Results Study enrollment will begin in 2022. Conclusions The vibrance-mg study will assess the PK/PD, safety and activity of Nipocalimab in pediatric gMG.

Vivacity MG Phase 3 Study: Clinical Trial of Nipocalimab Administered to Adults With Generalized Myasthenia Gravis

Objective We describe Vivacity-MG3. our pivotal Phase 3, Multicenter, Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Efficacy, Safety, Pharmacokinetics (PK), and Pharmacodynamics (PD) of Nipocalimab Administered to Adults With gMG (NCT04951622). Background Nipocalimab is a high affinity, fully human, aglycosylated, effectorless IgG1 anti FcRn monoclonal antibody that targets the neonatal Fc receptor (FcRn) with high affinity, thereby lowering IgG pathogenic antibodies in autoimmune disease. Data from Vivacity-MG, a Phase 2 randomized placebo-controlled study of nipocalimab in adult generalized myasthenia gravis (gMG), demonstrated safety, tolerability, and efficacy of nipocalimab (clinicaltrials.gov NCT03772587). Design/Methods This global study will enroll approximately 180 participants with gMG, aged 18 and older, with an insufficient clinical response to ongoing, stable standard-of-care therapy, as reflected by a Myasthenia Gravis-Activities of Daily Living (MG-ADL) score of >/= to 6 at screening and baseline, and a Myasthenia Gravis Foundation of America (MGFA) Class of IIa/b–IVa/b at screening. The study will consist of a screening period of up to 4 weeks, a 24-week double-blind placebo-controlled phase where participants will be randomly assigned in a 1:1 ratio to receive either placebo or nipocalimab intravenously every two weeks, and an open label extension phase of variable duration. The primary outcome is the average change in MG-ADL score from baseline to weeks 22, 23 and 24 of the double-blind placebo-controlled phase. Results Study enrollment began in July 2021 and is ongoing. Conclusions The ongoing Vivacity MG Phase 3 study will assess the efficacy, safety, and PK/PD of Nipocalimab in adult gMG.

DESMOSOMES IN ORAL DISEASES – A REVIEW

Desmosomes are the adhesion proteins that function both as an adhesive complex and as a cell surface attachment site for keratin intermediate filaments of the cytoskeleton. Desmosomes are more widely distributed along the lateral membranes. Desmosomes contain two types of transmembrane proteins desmogleins and desmocollins which belong to cadherin family. These desmosomal cadherins are linked to the keratin cytoskeleton via several cytoplasmic proteins such as desmoplakin and plakoglobin. Desmosomes play a critical role in the maintenance of normal tissue architecture. They are frequently mutated and desmosomal adhesion is compromised by antibodies in autoimmune diseases and result in blistering disorders in epithelium. Inherited mutations in genes in desmosomal constituents can also affect the skin and the heart. Desmosomes may have a tumour suppressor function and desmosomal cadherins have the capacity to suppress the invasiveness of cells in culture.

Correlation of enzyme-linked immunoassay with line immunoassay for the detection of antinuclear antibody in autoimmune diseases

INTRODUCTION: For the diagnosis of autoimmune disorders, detection of antinuclear antibodies (ANAs) is a key step. Indirect immunofluorescence assays are regarded as a gold standard method. Enzyme-linked immunosorbent assay (ELISA) is easily automated and less technical skills are needed and so used for ANA screening. Further testing is done with line immunoassay (LIA) for specific diagnosis of autoimmune disease. However, there is a lack of data comparing ELISA with LIA for ANA detection in the Indian population.OBJECTIVE: The objective of this study is to find the correlation between ELISA and LIA for ANA detection.METHODOLOGY: Serum samples received for ANA screen were tested using commercial kits for ELISA and LIA according to manufacturer's instructions.RESULTS: Total of 108 samples were tested both by ELISA and LIA. The total numbers of samples positive by ELISA were 26 and positive by LIA were 54 of 108. Samples that were both ELISA and LIA positive were 22 and both ELISA and LIA negative were 50. There was a fair level of agreement between the two methods (kappa = 0.333) with 95% confidence interval of 0.259–0.699. There was no significant difference in the distribution of most of the antibody types detected by LIA among ELISA positive and negative groups. However, the correlation of antibody type U1-Sn ribonucleoprotein with ELISA was found to be statistically significant (P = 0.0319).CONCLUSION: Detection of ANA is an important screening test. This present study shows moderate correlation between ELISA and LIA and hence both methods can be used depending on the workload of laboratories for cost-effectiveness. ELISA may be the preferred method for high-throughput laboratories.

Longitudinal profile of circulating T follicular helper lymphocytes parallels anti-HLA sensitization in renal transplant recipients.

Antibody-mediated rejection is responsible for 30%-50% of renal graft failures. Differentiation of B cells into antibody-producing plasmablasts depends on the collaboration of follicular helper T cells (Tfh). We analyzed circulating Tfh (cTfh) in kidney recipients and studied cTfh relationship with anti-HLA antibody production and graft outcome. cTfh were longitudinally analyzed in a prospective cohort of patients (n=206), pre- and posttransplantation. Clinical data, HLA sensitization, and cTfh function were recorded. Both pretransplant and 6-month posttransplant cTfh were able to derive IgG-producing plasmablasts. Pretransplant cTfh was decreased in patients, especially in those who received dialysis. However, these cells were increased in patients with previous allograft or transfusions and in HLA-sensitized recipients. After transplantation cTfh expanded, significantly more in patients who developed de novo anti-HLA antibodies than in patients who remained unsensitized. Augmented pretransplant cTfh positively correlated with higher intensity of pretransplant anti-HLA class I and with de novo anti-HLA class I and anti-HLA class II antibodies. Consistently, pretransplantation cTfh were higher in patients who experienced acute rejection (HR=1.14 [1.04-1.25]). Thus, we show a role for Tfh in anti-HLA sensitization and rejection. Multicenter studies with additional patient cohorts are needed to validate these results. Immunosuppressive drugs targeting Tfh could be useful to improve outcomes.

Plasma Cell Differentiation Pathways in Systemic Lupus Erythematosus.

Plasma cells (PCs) are responsible for the production of protective antibodies against infectious agents but they also produce pathogenic antibodies in autoimmune diseases, such as systemic lupus erythematosus (SLE). Traditionally, high affinity IgG autoantibodies are thought to arise through germinal center (GC) responses. However, class switching and somatic hypermutation can occur in extrafollicular (EF) locations, and this pathway has also been implicated in SLE. The pathway from which PCs originate may determine several characteristics, such as PC lifespan and sensitivity to therapeutics. Although both GC and EF responses have been implicated in SLE, we hypothesize that one of these pathways dominates in each individual patient and genetic risk factors may drive this predominance. While it will be important to distinguish polymorphisms that contribute to a GC-driven or EF B cell response to develop targeted treatments, the challenge will be not only to identify the differentiation pathway but the molecular mechanisms involved. In B cells, this task is complicated by the cross-talk between the B cell receptor, toll-like receptors (TLR), and cytokine signaling molecules, which contribute to both GC and EF responses. While risk variants that affect the function of dendritic cells and T follicular helper cells are likely to primarily influence GC responses, it will be important to discover whether some risk variants in the interferon and TLR pathways preferentially influence EF responses. Identifying the pathways of autoreactive PC differentiation in SLE may help us to understand patient heterogeneity and thereby guide precision therapy.

An anti-DNA antibody prefers damaged dsDNA over native

DNA–protein interactions, including DNA–antibody complexes, have both fundamental and practical significance. In particular, antibodies against double-stranded DNA play an important role in the pathogenesis of autoimmune diseases. Elucidation of structural mechanisms of an antigen recognition and interaction of anti-DNA antibodies provides a basis for understanding the role of DNA-containing immune complexes in human pathologies and for new treatments. Here we used Molecular Dynamic simulations of bimolecular complexes of a segment of dsDNA with a monoclonal anti-DNA antibody’s Fab-fragment to obtain detailed structural and physical characteristics of the dynamic intermolecular interactions. Using a computationally modified crystal structure of a Fab–DNA complex (PDB: 3VW3), we studied in silico equilibrium Molecular Dynamics of the Fab-fragment associated with two homologous dsDNA fragments, containing or not containing dimerized thymine, a product of DNA photodamage. The Fab-fragment interactions with the thymine dimer-containing DNA was thermodynamically more stable than with the native DNA. The amino acid residues constituting a paratope and the complementary nucleotide epitopes for both Fab–DNA constructs were identified. Stacking and electrostatic interactions were shown to play the main role in the antibody–dsDNA contacts, while hydrogen bonds were less significant. The aggregate of data show that the chemically modified dsDNA (containing a covalent thymine dimer) has a higher affinity toward the antibody and forms a stronger immune complex. These findings provide a mechanistic insight into formation and properties of the pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus, associated with skin photosensibilization and DNA photodamage.

Молекулярная динамика иммунного комплекса ДНК, содержащей фотоаддукт, с Fab-фрагментом антитела к ДНК

Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

Anti-PM/Scl antibodies are found in Japanese patients with various systemic autoimmune conditions besides myositis and scleroderma.

IntroductionAnti-PM/Scl antibodies are associated with polymyositis (PM)/systemic scleroderma (SSc) overlap syndromes and are also found in other systemic autoimmune diseases. Although anti-PM/Scl reactivity is found in 3-11% of PM or SSc patients and in approximately 25% of PM/SSc overlap patients, previous large studies of Japanese patients with scleroderma reported that anti-PM/Scl are not found in Japanese patients at all. The PM/Scl autoantigen complex comprises 11–16 different polypeptides; ELISA with PM1-α peptide, which is a major epitope of the PM/Scl complex, has frequently been used for the detection of these antibodies in recent studies. However, no ELISA kit is commercially available in Japan.MethodsIn this study, we developed an immunoassay for measuring antibodies against recombinant PM/Scl-100 and PM/Scl-75 polypeptides, which are the two major targets of the complex, and we investigated their presence in 600 Japanese patients with various systemic autoimmune conditions. Immunoprecipitation analysis using the recombinants in addition to traditional radiolabeled cell extracts were also applied to ELISA-positive sera.ResultsIn ELISA, 11 patients were positive for anti-PM/Scl-100 antibodies and 7 of these 11 patients were also positive for anti-PM/Scl-75 antibodies. Immunoprecipitation analysis using the recombinants in addition to traditional radiolabeled cell extracts confirmed that 9 out of these 11 patients immunoprecipitated the typical sets of PM/Scl proteins. In total, 4/16 (25%) undifferentiated connective tissue disease (UCTD) patients, 3/126 (2.4%) dermatomyositis patients, 1/223 (0.4%) SSc patients, 1/88 (1.1%) Sjögren’s syndrome patients, 0/123 patients with systemic lupus erythematosus, 0/17 patients with overlap syndrome and 0/7 patients with PM were judged to be positive for anti-PM/Scl antibodies.ConclusionsThis is the first report of Japanese autoimmune patients with anti-PM/Scl antibodies. In Japanese patients, anti-PM/Scl antibodies are only very rarely found, and they are not always specific for dermatomyositis (DM) or SSc; they are also present in various autoimmune conditions with the highest prevalence being in UCTD. All anti-PM/Scl-positive DM cases are complicated with interstitial lung disease and/or cancer, while no life-threatening involvement was found in other anti-PM/Scl-positive cases. Further studies on larger cohorts are necessary to define the clinical significance of anti-PM/Scl antibodies in autoimmune diseases.

Predictive value of antinuclear antibodies in autoimmune diseases classified by clinical criteria: Analytical study in a specialized health institute, one year follow-up

Introduction: Determination of antinuclear antibodies (ANA) by indirect immunofluorescence (IIF) is usually the initial test for the diagnosis of systemic rheumatic diseases (SRD). Assigning predictive values to positive and negative results of the test is vital because lack of knowledge about ANAs and their usefulness in classification criteria of SRD leads to inappropriate use. Methods: Retrospective study, ANA tests requested by different specialties, correlation to patients' final diagnosis. Results: The prevalence of autoimmune disease was relatively low in our population yielding a low PPV and a high NPV for the ANA test. 40% of the patients had no clinical criteria applied prior to test. Coexistence of two or more autoimmune disorders affects prevalence and predictive values. Conclusion: Application of the test after careful evaluation for clinical criteria remarkably improves the positive likelihood ratio for the diagnosis.

SAT0285 Progranulin-Neutralizing Autoantibodies in Psoriatic Arthritis

BackgroundPsoriatic arthritis (PsA) is a distinctive inflammatory arthritis that may develop in 5-40% of individuals suffering from psoriasis. We recently described progranulin autoantibodies (PGRN-Abs) in the sera of patients with...

Evaluation of an automated chemiluminescent immunoassay kit for antinuclear antibodies in autoimmune diseases

The identification of antinuclear antibodies (ANA) is an essential step in the diagnosis of different autoimmune diseases. The gold standard method for their detection is immunofluorescence assay. However, this is a subjective and laborious method, thus a need for simplified objective methods has aroused. In the current work, we evaluated such automated method, the LIAISON(®) (DiaSorin, Italy) for the detection of ANA. A total of 242 sera were analyzed including 67 from healthy subjects, 107 from primary biliary cirrhosis (PBC) patients, 20 from scleroderma patients and 48 from patients with Sjögren's syndrome. All sera were analyzed using the automated chemiluminescent immunoassays, LIAISON(®) for the presence of ANA (kit No. 310300). Positive samples were further analyzed for the presence of antidouble-stranded DNA (dsDNA) and autoantibodies to 6 extractable nuclear antigens (ENA) of the LIAISON(®) (kits No. 310330 and 310331). Negative samples were further analyzed by Blueblot ANA assay (D-TEK, Belgium) or BlueDot Liver (D-TEK, Belgium) as appropriate. The LIAISON(®) specificity for ANA screening was 97%. ANA positivity was determined in 80% of all patients. The sensitivity was 95.5% in scleroderma, 83% in PBC and 72.9% in Sjogren's syndrome. ENA was positive in all ANA-positive scleroderma and Sjögren's sera and in 27% of ANA-positive PBC sera. Among scleroderma or Sjögren patients that were ANA negative, 4 samples were positive for anti-SSA and 2 for RNP-68 utilizing Blueblot assays. M2 protein was found in 1 out of the ANA-negative PBC patients. The LIAISON(®) ANA screen is specific and sensitive for the evaluation of ANA in patients with primary biliary cirrhosis, scleroderma and Sjögren's syndrome.

Pulmonary thromboembolism in children with rheumatic diseases

Results Thrombotic events have been associated with antiphospholipid antibodies in autoimmune diseases, including systemic lupus erythematosus (SLE). However, pulmonary thromboembolism (PTE) from deep vein thromboses (DVT) or in situ pulmonary arterial thrombosis is uncommon in rheumatic diseases, especially in children. The diagnosis and treatment of PTE may be delayed due to a paucity of symptoms or to symptoms attributed to more common manifestations such as pleuritis or pneumonia. We report findings in 6 children with PTE secondary to SLE (4), Systemic Sclerosis (SSc) (1) and Polyarteritis Nodosa (PAN) (1).

Anti-Idiotypic Antibodies and “Tumor-Only” Antigens: An Update

The use of anti-idiotypic antibodies for cancer treatment continues to be a major field of investigation. One ma- jor challenge for tumor immunotherapy is the identification of antigens associated only or preferably with malignant cells. We summarize here some of the most recent preclinical and clinical advances using two targets that can be considered tumor-specific antigens: N-glycolyl (NeuGc)-gangliosides and the idiotype of B cell lymphomas. Recent developments with tumor-associated protein antigens and the role of anti-idiotypic antibodies in autoimmune diseases are also discussed.

Increased expression of Matrix Metalloproteinase 9 in liver from NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein

BackgroundHuman parvovirus B19 infection has been postulated to the anti-phospholipid syndrome (APS) in autoimmunity. However, the influence of anti-B19-VP1u antibody in autoimmune diseases is still obscure.MethodsTo elucidate the effect of anti-B19-VP1u antibodies in systemic lupus erythematosus (SLE), passive transfer of rabbit anti-B19-VP1u IgG was injected intravenously into NZB/W F1 mice.ResultsSignificant reduction of platelet count and prolonged thrombocytopenia time were detected in anti-B19-VP1u IgG group as compared to other groups, whereas significant increases of anti-B19-VP1u, anti-phospholipid (APhL), and anti-double strand DNA (dsDNA) antibody binding activity were detected in anti-B19-VP1u group. Additionally, significant increases of matrix metalloproteinase-9 (MMP9) activity and protein expression were detected in B19-VP1u IgG group. Notably, phosphatidylinositol 3-phosphate kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (ERK) proteins were involved in the induction of MMP9.ConclusionThese experimental results firstly demonstrated the aggravated effects of anti-B19-VP1u antibody in disease activity of SLE.

B Cell Targeting Therapy Using the Anti-CD20 Antibody in Autoimmune Diseases

Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are representative autoimmune diseases thought to involve disturbances in T and B cell functions. Immune complexes consisting of antigens and autoantibodies secreted from activated B cells cause severe inflammation in various organs. Since often patients with RA and SLE are refractory to these conventional treatments such as immunosuppressants and corticosteroids, innovative approaches need to be developed. CD20 is a surface molecule specific for B cells and rituximab is a chimeric antibody specific for human CD20 and is known to deplete B cells. Recently, the potential efficacy of B-cell depletion therapy with rituximab has been reported in several autoimmune diseases. Rituximab is now approved for use in combination with methotrexate in refractory RA patients in the United States and EU. We reported that SLE patients with organ-threatening disorders that are resistant to intensive conventional therapies, were treated by once a week administration of anti-CD20 antibody rituximab, and that sufficient evidence concerning the excellent tolerability and high efficacy of rituximab therapy was obtained in both a pilot study and a nation-wide phase I/II clinical examination Moreover, a rapid and marked reduction in the expression of the co-stimulatory molecules CD40 and CD80 on B-cells was found in SLE patients, implying that reduction of both the quantity and the quality of B-cells by rituximab could improve the disease course in refractory SLE. Therefore, targeting B-cells may have potential interests by bringing about a breakthrough in the treatment of RA, SLE and other autoimmune diseases.

The autoimmune diseases manifested by production of autoantibodies: the autoantigens identified by random peptide library.

Phage-displayed random peptide libraries (RPL) provide a powerful technique for identification, structural and functional analysis of ligands for many different target molecules, including, antibodies, receptors or other proteins. This strategy has been verified to be an effective tool for research in immunology and successfully has been used to determine the target sequence for monoclonal and polyclonal antibodies. The peptide library approach provides great promise for characterization of ligands with no prior information concerning antibody specificity. This would allow the recognition of candidate antigens involved in initiation or perpetuation of autoimmune diseases. This technology also offers the potential for new therapeutic opportunities, production of diagnostic reagents, or even development of effective new vaccines. This review focuses on studies regarding the identification of autoantigens recognized by antibodies in autoimmune diseases using phage-display peptide libraries.

Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models

BackgroundPhage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated.ResultsWe were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". These are produced by genetic fusion of anti-tTG scFv to Human, Mouse or Rat Fc domains, making them suitable for in vivo expression. The results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous Fc domains. Further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. Finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host.ConclusionIn this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of Celiac Disease. This can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases.

Complete DNA Sequence Variation in the Apolipoprotein H (β2‐glycoprotein I) Gene and Identification of Informative SNPs

Apolipoprotein H (APOH), also known as beta2-glycoprotein I, is a major antigen for the production of antiphospholipid antibodies in autoimmune diseases. Previously we have examined DNA variation in the coding region of the APOH gene and determined the molecular basis of the common protein polymorphism. Here we report the results of DNA sequence variation in the entire APOH gene encompassing a 20.3 kb region in 46 Caucasian Americans and 48 African American chromosomes. A total of 150 single nucleotide polymorphisms (SNPs) and one tri-allelic polymorphism were identified, including 8 in the coding region, 14 in the 5'-region and 2 in the 3'- region; the remainder were observed in introns. The observed number of SNPs was higher in the African American sample than in the Caucasian sample (130 vs. 84). We examined the race-specific linkage disequilibrium pattern among SNPs and identified maximally informative SNPs for future association studies. Altogether, we have identified 17 informative SNPs among Caucasians and 35 in blacks. The discovery of a full range of sequence variation and identification of race-specific informative SNPs in the APOH gene may facilitate the rapid evaluation of this variation in relation to autoimmune diseases.

The Prevalence of Anti-acetylcholinesterase Antibodies in Autoimmune Disease

A robust and precise enzyme linked immunosorbent assay (ELISA) with proven sensitivity and specificity has been employed to detect human antibodies (allogenic/autogenic) to human acetylcholinesterase (AChE). The sensitivity of the method has been established using mouse monoclonal antibodies (0.8 ng/ml) and uniquely, human sera positive for anti-Yta allogenic antibodies, to one phenotypic form (most common) of human AChE. The latter was also used as the positive human control to ensure functionality of the assay. The ELISA method was used to establish a normal distribution curve for absorbance values employing sera from healthy blood donors Subsequently, the ELISA was employed to investigate the prevalence of anti-AChE antibodies in patients with confirmed autoimmune disease and patients with non-autoimmune thyroid disease (diseased control). The results indicate that there is not a high prevalence of anti-AChE antibodies in patients with confirmed autoimmune disease. The lack of anti-AChE autoantibodies in patients' with clinically apparent Graves' ophthalmopathy, mitigates against there being a causal role of such antibodies in Graves' associated eye disease.