A Cell-Based Assay for Detection of Anti-Fibrillarin Autoantibodies in Systemic Sclerosis

Objectives Anti-fibrillarin antibodies are useful for establishing diagnosis and predicting distinct clinical features in systemic sclerosis (SSc). Anti-fibrillarin produces a characteristic clumpy nucleolar pattern in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) that is useful to guide further testing in antigen-specific immunoassays (ASI). Immunoprecipitation (IP) is the gold standard ASI for anti-fibrillarin determination. We established a new anti-fibrillarin Cell-Based Assay (CBA) and compared its diagnostic performance with IP and ASI commercial kits. The clinical features of SSc patients with and without anti-fibrillarin antibodies were analyzed. Methods A TransMembrane Signal (TMS) was added to the human fibrillarin gene in order to drive the transgenic fibrillarin to the cytoplasmic membrane. HEp-2 cells transfected with a vector containing the TMS-fibrillarin were used as substrate for IFA in the CBA. Sixty-two samples with high-titer nucleolar pattern in HEp-2 IFA (41 clumpy and 21 homogeneous/punctate) were tested for anti-fibrillarin in the CBA, IP, line-blot and ELISA. Clinical SSc phenotype was evaluated in 106 patients according to positive/negative anti-fibrillarin result in the CBA. Results TMS-fibrillarin was properly located to the cytoplasmic membrane and recognized by human autoantibodies. Thirty-eight of 41 clumpy nucleolar samples (92.7%) and none of 21 samples with other nucleolar patterns were positive for anti-fibrillarin in the CBA. There was 100% agreement between the positive/negative results in the CBA and IP. Among the 38 CBA-positive samples, only 15 (39.5%) and 11 (29%) were considered positive for anti-fibrillarin in the line-blot and ELISA, respectively. Anti-fibrillarin was associated with higher frequency of diffuse cutaneous SSc (dcSSc) phenotype (72.7% vs 36.8%; p=0.022), cardiac involvement (36.4% vs 6.5%; p=0.001) and scleroderma renal crisis (18.2% vs 3.3% p = 0.028). Conclusion With an innovative strategy of targeting the transgenic autoantigen to the cell membrane, we developed a new straightforward assay for detection of anti-fibrillarin autoantibodies. This new CBA presented high sensitivity and specificity for the detection of anti-fibrillarin autoantibody, comparable to the gold standard IP. Moreover, anti-fibrillarin antibodies detected in the CBA identified patients with a higher frequency of dcSSc, cardiac and renal involvement. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by Sao Paulo Government agency FAPESP (Sao Paulo State Research Foundation) grant numbers #2017/20745-1 and #2021/04588-9. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The research was approved by the Ethics Committee of the Federal University of Sao Paulo (UNIFESP). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.