Development of ELISAs for the detection of novel myositis autoantibodies (TIF1 alpha, TIF beta and PUF60) in UKMyoNet patients - June 2018

Lay Summary

Myositis is a rare autoimmune condition hallmarked by muscle inflammation, weakness, a characteristic skin rash, and in some cases associated cancer and lung disease. Due to the wide range of presenting features the diagnosis of myositis can be problematic; however the screening of myositis autoantibodies is increasingly viewed as a key tool in diagnosis. 

At the University of Bath we have now established a clinical service screening patient blood samples for myositis autoantibodies using the ‘gold standard’ technique termed radioimmunoprecipitation (IPP). Through this service and with our continuing collaborations with the UKMyoNet, EuMyonet and Juvenile Dermatomyositis Cohort Biomarker Studies we have now screened over 3000 myositis patients for autoantibodies using IPP. 

These results, alongside the corresponding clinical data have now been extensively analysed in collaboration with the Department of Mathematics, and a manuscript detailing the percentage of myositis patients with each particular autoantibody, the number of cases with more than one type of autoantibody and the clinical features associated with each specific autoantibody is now in preparation for submission. 

Additionally, an abstract regarding the mutual exclusivity of myositis specific autoantibodies has been accepted as a poster presentation at the British Society of Rheumatology conference (April 2016) and work from this project has been presented as an oral presentation at the 1st International Myositis meeting (May 2015). This work has also led to a review in the Journal of Internal Medicine and a chapter in ‘The Myositis Handbook: An Inclusive Guide to the Inflammatory Myopathies’. Data from this study has also been used in additional publications including an analysis of the autoantibodies in the Hungarian cohort and a study of HMGCR autoantibodies in patients from the Czech Republic. 

Additionally, work has been completed on a revised manuscript regarding the novel autoantibody; anti-EIF3 in patients with mild polymyositis, and the continued development of rapid tests for determining the levels of anti-TIF1 gamma, anti-NXP2 and anti-MDA5 autoantibodies. Furthermore, with the aid of two final year Pharmacology Students, preliminary work has been completed in the development of tests for anti-TIF1 alpha and beta autoantibodies, a study due to be continued as part of a BIRD studentship in the Summer (2016). 

Furthermore as part of a Medical Research Council grant we have been able to complete preliminary work investigating the accuracy of a commercially available myositis autoantibody assay in comparison with our IPP data, as well as collaborating with a new industrial partner (Genalyte) in the development and validation of rapid myositis test. 

The preliminary results from this study have been presented by Prof Neil McHugh at the American College of Rheumatology meeting and will form the basis of a grant application to ‘The Myositis Association’ investigating the standardisation of commercial myositis autoantibody tests. Additionally, the line blot versus IPP data is currently being analysed and prepared for submission. Bath Institute for Rheumatic Diseases Supporting Patients through Education & Research Page 2 of 2 Scleroderma Autoantibody Studies Our publication on the novel anti-EIF2B autoantibody, associated with diffuse systemic sclerosis and lung disease, has been accepted by Arthritis and Rheumatology. Work is now on going determining the presence of this autoantibody in a US cohort. Autoantibodies in Lung Disease. 

Finally, as part of a collaboration with the University of Liverpool, 250 idiopathic pulmonary fibrosis (IPF) patients have been screened for autoantibodies by IPP, with 2% resulting in a known autoantibody and a further 40% of patients demonstrating strong bands of unknown specificity, (some of which are now being investigated further as potentially novel lung disease markers). Since patients with IPF have a poor prognosis, the detection of autoantibodies, demonstrating a potential misdiagnosis, has clinical relevance. These preliminary findings are forming part of an MRC Partnership Grant application investigating the use of autoantibody screening as part of IPF / CTD diagnosis in a larger cohort of lung disease patients.

Scientific Summary

The University of Bath research group is well regarded as one of the leading centres for myositis serology research, and in the last 12 months has established a clinical service for autoantibody testing. Through this new service and our continued collaboration with the UKMyoNet, EuMyonet and Juvenile Dermatomyositis Cohort Biomarker Study networks we have expanded our myositis serum biobank to nearly 3000 myositis samples. 

All of these samples have been screened for myositis specific and associated autoantibodies by immunoprecipitation and work has been finalised verifying the results (as appropriate) by either repeat immunoprecipitations, in house ELISAs or using commercial assays. Where applicable, the serological and clinical data for these patients has been fully analysed with the assistance of the Department of Mathematics (University of Bath) and a manuscript describing the clinical associations of each myositis autoantibody, their mutual exclusivity and myositis specificity is in preparation. 

Additionally, an abstract regarding the mutual exclusivity of myositis specific autoantibodies (MSAs) has been accepted as a poster presentation at the British Society of Rheumatology annual conference (April 2016) and work from this project has been presented as an oral presentation at the 1st International Myositis meeting (Stockholm May 2015). This work has also led to two invited reviews / book chapters on the clinical and diagnostic utility of myositis autoantibodies: 

  • Myositis-specific autoantibodies: an important tool to support diagnosis of myositis. Betteridge Z, McHugh N. J Intern Med. 2016 Jul;280(1):8-23. Epub 2015 Nov 25. 
  • Other Myositis-associated and Myositis-Specific Autoantibodies. Z Betteridge. The Myositis Handbook: An Inclusive Guide to the Inflammatory Myopathies. Epub 2016 

Data from this study has also been included in a number of associated studies by collaborating groups across Europe and has formed part of a number of publications: 

  • Increasing incidence of immune-mediated necrotizing myopathy: single-centre experience. Klein M, Mann H, Pleštilová L, Zámečník J, Betteridge Z, McHugh N, Vencovský J.Rheumatology (Oxford). 2015 Nov;54(11):2010-4. doi: 10.1093/rheumatology/kev229. Epub 2015 Jun 24. 
  • Rare myositis-specific autoantibody associations among Hungarian patients with idiopathic inflammatory myopathy. Bodoki L, Nagy-Vincze M, Griger Z, Betteridge Z, Szöllősi L, Jobanputra R, Dankó K. Acta Reumatol Port. 2015 OctDec;40(4):337-347. 
  • [Comparison of clinical characteristics and laboratory parameters of patients with dermatomyositis-specific autoantibodies and autoantibody-negative patients]. Bodoki L, Budai D, Nagy-Vincze M, Griger Z, Betteridge Z, Dankó K. Orv Hetil. 2015 Sep 6;156(36):1451-9. Hungarian. 

Furthermore, this myositis serology study has led to the development of a number of substudies as detailed below: 

Anti-EIF3  

The discovery of a novel autoantibody (anti-EIF3) in patients with mild polymyositis has been previously presented as oral and poster abstracts at a number of meetings. A manuscript describing the findings was submitted to Arthritis and Rheumatology but was rejected after review. In the past 12 months, the reviewers’ comments have been addressed and a revised manuscript has been prepared for submission to Rheumatology. The project is now awaiting additional serum from the anti-EIF3 positive patients in order to complete the final figures for the paper. 

Validation of Commercial Assays 

In the past year we have initiated a collaboration with a US diagnostic’s company ‘Genalyte’. This company has developed a rapid autoanalyser (the Maverick System) capable of multiplex screening of samples in less than 20 minutes. Having been provided one of these autoanalysers our group has been testing the myositis autoantibody panels using samples from our vast serum bank. In addition, we have also been screening the same samples on the commercially available EuroImmun blotting panel and comparing the data. The preliminary results have been presented by Prof Neil McHugh at the American College of Rheumatology annual meeting and will form the basis of a grant application to ‘The Myositis Association’ investigating the validation and standardisation of commercial myositis autoantibody tests. Additionally, the lineblot versus immunoprecipitation data is currently being analysed and prepared for submission both as a meeting abstract and a full manuscript. 

ELISA Development (TIF1, NXP2, MDA5) 

We previously developed in-house ELISAs for the detection of NXP2, MDA5 and TIF1- gamma. These ELISAs have been used to screen for, and confirm the presence of, anti-NXP2, anti-MDA5 and anti-TIF1 gamma in the myositis and control cohorts. Additionally, the ELISAs have been enhanced to be a quantitative assay, and serial samples from autoantibody positive patients have been screened to determine whether the autoantibody levels vary over time and if this has any correlation with clinical symptoms. 

In addition, with the assistance of two University of Bath final year pharmacology undergraduates preliminary work in establishing ELISAs and western blots for the detection of TIF1 alpha and beta has been initiated. These initial findings have proven the feasibility of establishing these assays within the lab and a BIRD summer studentship has been appointed to undertake this work in August and September 2016. 

Scleroderma Autoantibody Studies 

After completion of the additional work requested by reviewers, our publication on the novel scleroderma specific anti-EIF2B autoantibody has been accepted by Arthritis and Rheumatology as a concise report. Additional work on this topic is now underway by the group (Miss Hui Lu, Dr Zoe Betteridge, Dr John Pauling and Prof Neil McHugh) investigating the presence of this autoantibody in a US Systemic Sclerosis cohort 

  • Anti-Eukaryotic Initiation Factor 2B Autoantibodies are Associated with Interstitial Lung Disease in Patients with Systemic Sclerosis. Betteridge ZE, Woodhead F, Lu H, Shaddick G, Bunn CC, Denton CP, Abraham DJ, du Bois RM, Lewis M, Wells AU, McHugh NJ. Arthritis Rheumatol. 2016 

Autoantibodies in Lung Disease As part of a collaboration with the University of Liverpool 250 idiopathic pulmonary fibrosis (IPF) patients have been screened for autoantibodies by immunoprecipitation with approximately 2% of patients resulting in a known connective tissue disease (CTD) autoantibody and a further 40% of patients demonstrating strong bands of unknown specificity. Since patients with IPF have a poor prognosis with limited treatment options, the detection of CTD autoantibodies, demonstrating a potential misdiagnosis, in a subset of these patients has clinical relevance. These preliminary findings are now been used as part of an MRC Partnership Grant application to investigate the use of autoantibody screening as part of IPF / CTD diagnosis in a larger cohort of lung disease patients. 

Additionally, since 4% of patients in the cohort resulted in the same novel pattern on immunoprecipitation, work is now being completed investigating this potential novel lung disease specific autoantibody.