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

TY - JOUR

T1 - Validation of an indirect ELISA assay for assessment of autoantibodies against full-length TRIM21 and its individual domains

AU - Dahl, Marie Louise Næstholt

AU - Mikkelsen, Jakob Hauge

AU - Hvid, Malene

AU - Korsholm, Trine-Line

AU - Nielsen, Kirstine Overgaard

AU - Andersen, Christian Brix Folsted

AU - Greisen, Stinne

AU - Deleuran, Bent

PY - 2023/9

Y1 - 2023/9

N2 - 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.

AB - 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.

KW - Humans

KW - Autoantibodies

KW - Sjogren's Syndrome/diagnosis

KW - Lupus Erythematosus, Systemic/diagnosis

KW - Autoantigens

KW - Protein Domains

KW - Enzyme-Linked Immunosorbent Assay

U2 - 10.1080/00365513.2023.2221862

DO - 10.1080/00365513.2023.2221862

M3 - Journal article

C2 - 37379227

SN - 0085-591X

VL - 83

SP - 309

EP - 317

JO - Scandinavian Journal of Clinical and Laboratory Investigation

JF - Scandinavian Journal of Clinical and Laboratory Investigation

IS - 5

ER -