Immunoblot assay matches ELISA performance for autoimmune hepatitis diagnosis
Italian researchers have demonstrated that immunoblot testing for anti-F-actin antibodies delivers diagnostic accuracy equivalent to established ELISA methods in autoimmune hepatitis type 1. The study reveals comparable specificity and overall performance metrics, potentially offering laboratories enhanced diagnostic flexibility for this rare but serious liver condition.
Researchers at the University of Padova have unveiled promising evidence that immunoblot assays could serve as a reliable alternative to enzyme-linked immunosorbent assay (ELISA) testing for detecting anti-F-actin antibodies in autoimmune hepatitis type 1 (AIH-1), a rare inflammatory liver disease affecting approximately 19 per 100,000 people in Europe.
Comparable accuracy achieved across testing platforms
The study, published in LabMed Discovery on 12 August 2025, analysed 86 serum samples from three distinct patient groups: 14 with confirmed AIH-1, 38 with other liver disorders, and 34 with unrelated non-liver conditions. Both testing methods demostrated identical diagnostic accuracy of 81.4%, though with differing performance characteristics.
Lead researcher Giulia Musso and colleagues found that whilst immunoblotting showed lower sensitivity than ELISA (50% versus 78.6%), it compensated with superior specificity (87.5% versus 81.9%). “The diagnostic accuracy of (true positive + true negative)/total number of patients was 81.4% for both the immunoblot and ELISA,” the authors reported.
Receiver operating characteristic analysis revealed no statistically significant difference between the methods, with area under curve values of 0.77 for immunoblotting and 0.82 for ELISA.
Enhanced specificity offers diagnostic advantages
The improved specificity of immunoblot testing addresses a key clinical challenge in AIH-1 diagnosis. Traditional smooth muscle antibody (SMA) testing using indirect immunofluorescence lacks disease specificity, as these antibodies can appear in various infectious, rheumatological, and other hepatological disorders.
“Immunoblotting might be a reliable assay for the identification of anti-F-actin antibodies, and given its high specificity, its implementation in a clinical laboratory might confirm the specific diagnosis of AIH-1 in patients with IIF-detected SMA,” the researchers concluded.
Multiplexed testing capabilities enhance laboratory efficiency
A significant advantage of immunoblot assays lies in their capacity for simultaneous multi-antibody testing. The European Autoimmune Liver Diseases panel used in this study can detect multiple liver-related autoantibodies including anti-mitochondrial antibodies, liver-kidney microsomal antibodies, and soluble liver antigen antibodies in a single assay.
This multiplexed approach “allows simultaneous testing of multiple specific antibodies other than F-actin,” potentially confirming diagnoses of different autoimmune liver diseases in patients presenting with challenging clinical indications, the authors noted.
Clinical implications and implementation considerations
The research highlights distinct advantages for each testing approach. While ELISA remains superior for monitoring antibody concentrations during patient follow-up, immunoblot testing offers practical benefits for laboratories with limited autoimmune testing workloads, requiring no calibration curves and providing single-sample testing capability.
However, the authors acknowledged several study limitations, including the small sample size and retrospective design. “Given its lower sensitivity compared to ELISA, highly suspected false negative results should be confirmed with other immunoassays that offer higher sensitivity,” they cautioned.
Future research directions
The study represents the first direct comparison between immunoblot and ELISA methods for anti-F-actin antibody detection. The researchers emphasised that larger prospective studies are necessary before routine clinical implementation, particularly given that current guidelines do not recommend using anti-F-actin solid-phase assays alone for AIH screening.
Early and precise AIH-1 diagnosis remains crucial, as untreated disease typically progresses to liver failure with most patients dying within five years. Timely immunosuppressive therapy can prevent disease progression and significantly improve survival rates.
Reference: Musso, G., Gallo, N., & Basso, D. (2025). Diagnostic performance of an immunoblot assay for anti-F-actin antibodies in type 1 autoimmune hepatitis. LabMed Discovery, 2, 100086.
https://doi.org/10.1016/j.lmd.2025.100086
