Validation of an indirect ELISA assay for assessment of autoantibodies against full-length TRIM21 and its individual domains

Abstract

Anti-SSA-autoantibodies are common in patients with rheumatologic disease, especially Sjögren’s syndrome, systemic lupus erythematosus and rheumatoid arthritis. They consist of both autoantibodies towards Ro60 and Ro52, the latter also known as TRIM21. TRIM21 is an intracellular protein consisting of four domains; PRY/SPRY, Coiled-Coil, B-box and RING. The aim of this study was to establish an indirect ELISA detecting autoantibodies towards both the full-length TRIM21 protein and its four domains. We expressed the five constructs, created, and validated indirect ELISA protocols for each target using plasma from anti-SSA positive patients and healthy controls. Our findings were validated to the clinically used standards. We measured significantly higher levels of autoantibodies towards our full-length TRIM21, and the PRY/SPRY, Coiled-Coil and RING domains in patients compared to healthy controls. No significant difference in the level of autoantibodies were detected against the B-box domain. Our setups had a signal to noise ratio in the range of 30 to 184, and an OD between 2 and 3. Readings did not decline using NaCl of 500 mM as wash, affirming the high binding affinity of the autoantibodies measured. Our protocols allow us to further study the different autoantibodies of anti-SSA positive patients. This creates the possibility to stratify our patients into subgroups regarding autoantibody profile and specific pheno- or endotype.

Keywords:

• TRIM21
• autoantibodies
• anti-Ro52-autoantibodies
• anti-SSA-autoantibodies
• autoimmune disease
• Sjögren’s syndrome
• congenital AV-block
• rheumatology
• ELISA validation
• indirect ELISA

Acknowledgements

The authors thank everyone in Deleuran Lab, Aarhus University for laboratory assistance and general discussions on the project. Furthermore, we wish to thank the staff at the Department of Rheumatology, Aarhus University Hospital for their assistance in finding possible project subjects and inclusion of patient samples.

Disclosure statement

No potential conflict of interest was reported by the authors.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

Dagmar Marshalls Fond: No nr available; Fonden til Lægevidenskabens Fremme: L-2021-00148; Grosserer L. F. Foghts Fond: 22.057; Gigtforeningen: R203-A7197.