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More than 15 autoantibodies have been identified in patients with idiopathic inflammatory myopathies (IIM). Most of them are specific to patients with IIM and therefore called myositis-specific autoantibodies (MSA).1 There is no standardised approach of MSA testing to be used in clinical settings. Immunoprecipitation (IP) of RNA and/or proteins in cellular lysates has traditionally been considered the golden standard for most autoantibodies but does not differentiate between antibodies targeting proteins with the same molecular weight and is not routinely available. Line blot assays (LB) represent a faster and semi-quantitative option to detect autoantibodies.2 3 Our aim was to compare a LB version with 16 specificities and an IP-based algorithm, as well as to describe clinical associations to autoantibody specificities in a cohort of patients classified as IIM.
The first available sera collected between 2013 and 2017, from 110 patients classified as having IIM4 followed at the rheumatology clinic at Karolinska University Hospital (Stockholm, Sweden) and sera from 60 healthy controls were included. We used a commercial LB assay (Euroline Myositis Antigen Profile 4, Euroimmun, Lübeck, Germany) with densitometrical evaluation of staining intensity for 16 IgG autoantibodies at the Department of Clinical Immunology, Uppsala University Hospital. The results were reported as negative or positive (0–10 vs ≥11 densitometry units) according to the manufacturer’s instructions. For the IP protocol, samples were analysed by protein-IP and RNA- IP at Kyoto University.5 6 When these results suggested antibodies targeting a 140kD protein, an in-house anti-MDA5 ELISA was performed (online Supplementary data)). Categorical variables were analysed by χ2 or Fisher’s test. Agreement was calculated by Cohen’s kappa.7
Results
The frequency of any autoantibody was similar by both assays (LB 48/111=43%; IP 45/111=41%, p=0.79). The most frequent MSAs were anti-Jo-1 (LB: 16%, IP: 10%) and anti-TIF1γ (LB: 7%, IP: 11%) followed by anti-Mi-2 α or β (LB: 7% IP: 2%), anti-SAE1 (LB: 4% IP:3%) and anti-SRP (LB: 3% IP: 4%). Patients with inclusion body myositis (IBM) and controls were negative for Jo-1, PL-7, SRP, MDA5 and TIF1γ specificities in all assays. Three controls had either anti-Ku, anti-NXP2 or anti-Mi-2α by LB (mean 20 units), corresponding to a total diagnostic specificity of 99.7% (table 1).
Frequencies of autoantibodies detected by LB and IP assays across EULAR/ACR myositis subtypes
The overall concordance rate between the two assays was 78% with moderate agreement (κ: 0.54). We found very good agreement for anti-SRP (k: 0.85), anti-Ku (κ:0.85) and anti-SAE1 (κ:0.85) and good agreement for anti-Jo-1 (κ:0.69). Agreement for anti-PmScl (κ:0.48), anti-MDA5 (κ:0.49), and anti-TIF1γ (κ:0.56) was moderate (table 2).
Comparisons between immunoprecipitation and line blot assays in 110 patients with inflammatory myopathies according EULAR/ACR criteria
Anti-Jo-1 autoantibodies were detected in 18 sera, 11 were LB+/IP+, 7 were LB+/IP- and 1 was LB-/IP+. Sera from five LB+/IP- patients were also evaluated by a commercial anti-Jo1 ELISA (Profile, Phadia, Freiburg, Germany): three were positive, one borderline and one negative. Anti-Jo-1 LB+ patients had a higher frequency of interstitial lung disease (p<0.001) and arthritis (p<0.001) compared with anti-Jo-1 negative patients (Table S1). Nineteen per cent of DM/PM patients were Jo-1+ compared with 0% of IBM (p<0.001). Anti-TIF1γ was detected in 14 patients; 6 patients were LB+/IP+ (43%), 2 were LB+/IP- (14%) and 6 were LB-/IP+ (43%). Among anti-TIF1γ+patients, by LB 63% and by IP 58% had cancer-associated myositis (Table S1 and S2). Anti-SAE1 autoantibodies were detected in four patients by LB, and three of these sera immunoprecipitated two 40 kDa and 90 kDa proteins, consistent with the two subunits of the heterodimer small-ubiquitin-like modifier activating enzyme (SAE1/SAE2) suggesting IP reactivity to SAE in 75% of the LB+ samples.8 Anti-Mi2 autoantibodies were more prevalent by LB than by IP. A plausible explanation is that the antigens are different between the two methods. The LB applied in our study included both alpha and beta proteins of the Mi2 protein, whether this affects the specificity could not be addressed in our study. However, our result is consistent with a previous report demonstrating positive anti-Mi2 autoantibodies in PM patients in European populations.9
Supplemental material
In conclusion, the concordance rate between the LB and IP assays for detection of MSA in patients classified as IIM according to the European League Against Rheumatism/American College of Rheumatology criteria was moderate for the most prevalent MSA. The low sensitivity for anti-TIF1 gamma antibody by LB is of concern, as this is a marker of cancer-associated DM. LB assay seems to be valid and useful to identify subgroups of IIM with specific clinical features.
Supplemental material
Footnotes
Handling editor Josef S Smolen
Contributors Study concept and design: IEL, JR. Acquisition of data: JR, HS, TM, FE. Statistical analysis: FE. Analysis and interpretation of data, drafting manuscript and critical revision of manuscript: all authors.
Funding The Swedish Research Council and King Gustav V:th 80-year Foundation.
Competing interests IEL has received honoraria from Bristol Myers Squibb and MedImmune and is currentlyreceiving a research grant from Bristol Myers Squibb and from Astra Zeneca; and is a scientificadvisor for Bristol Myers Squibb, aTyr and IDERA.
Patient consent for publication Not required.
Ethics approval Our study had the permit for antibody analysis from the Ethics Committee in Stockholm, Sweden.
Provenance and peer review Not commissioned; externally peer reviewed.