Semiquantitative analysis of line blot assay for myositis-specific and myositis-associated antibodies: a better performance?

Abstract

Semiquantitative analysis of line blot assay for myositis-specific and myositis-associated antibodies: a better performance? We read with interest the letter of Espinosa-Ortega F et al 1 comparing line immunoblot assay (LIA) and immunoprecipitation (IP) performance in the detection of myositis-specific antibody (MSA) and myositis-associated antibody (MAA). The authors found a global moderate agreement between tests, with a very good agreement for anti-signal recognition particle (SRP), anti-Ku and anti-small ubiquitin-like modifier activating enzyme (SAE1); a good agreement for anti-hystidil-tRNA synthetase (Jo-1) and a moderate agreement for anti-PM/Scl, anti-melanoma differentiation-associated gene 5 (MDA5) and anti-transcription intermediary factor 1 (TIF1 gamma). We previously analysed the performance of LIA (Euroline Autoimmune Inflammatory Myopathies 15 Ag (IgG) Euroimmun, Lubeck, Germany) and IP, revealing a lower global agreement (k:0.30), and different rate of agreement for single specificities, 2 compared with Espinosa-Ortega observation. In particular, we found a lower agreement rate for anti-Jo-1, PM/Scl and anti-Ku and higher agreement rate for anti-MDA-5, anti-TIF1 gamma and anti-Mi-2. Although the nature of antigens was the same (ie, native for Jo-1 and recombinant for others), we tried to find a reason for such discrepancies, separately analysing the concordance basing on the semiquantitative level of positivities of LIA. Considering only high positive (+++) values, we obtained a better agreement for anti-Mi-2 antibodies (from global k: 0.50 to high-positive k: 0.79) and anti-MDA-5 (from global k:0.63 to high-positive k:0.85). No difference of agreement rate was observed between tests if considering only high or moderate/ high positivities by LIA for anti-Jo-1, anti-nuclear matrix protein (NXP-2) and anti-TIF1 gamma. No comparisons have been made for non-Jo-1 anti-synthetases antibodies, anti-SRP and anti-SAE due to low number of positive sera. The introduction of commercial LIA improved the diagnostic accuracy of myositis patients, given that the routine use of IP is very hard in clinical practice. Anyway, some discrepancies between laboratories still remains, maybe due to the known lack of analyte-specific controls and calibrators with a possible inter-manufacturers variability. 3 Maybe, considering only high or moderate positivities could help the clinician to avoid false positive, in particular when multiple MSA specificities have been detected, considered a rare event in inflammatory myositides. 4

Double anti-PL-7 and anti-MDA-5 positive Amyopathic Dermatomyositis with rapidly progressive interstitial lung disease in a Hispanic patient

“…This was repeated later to ensure no false positivity, and results remained positive at similar levels for each antibody. According to the study done by Cavazzana et.al, there was lower agreement rate for anti-Jo antibodies, and higher agreement rate for anti-MDA-5 antibodies when analyzing the performance of immunoblot assay and immunoprecipitation, but there has been no comparison between these 2 methods for Anti-PL-7 antibodies [8].…”

Correspondence on ‘Standardisation of myositis-specific antibodies: where are we today?’

“…Only recently have real-life studies on larger series of patients and using a larger panel of MSA-related antigens been published, highlighting on one side a great intra-method analytical variability of DIA/LIA in detecting MSA,5 and on the other side their weak correlation with IP 6. Considering the controversial data between Espinosa-Ortega’s1 and Cavazzana’s7 8 studies, it is emblematic that there is still no concordance among LIA and IP even for anti-Jo1, the most common MSA and the first discovered in this group of diseases. The low agreement of IP versus other methods, evidenced by recent studies,2 6 7 9 raises the question of whether IP should still be considered the reference method for detecting MSA.…”

“…Prospective and multicentre studies are needed to validate these new methods and clarify whether they can be reliably used instead of the reference IP method. 15 Aggarwal et al 16 Infantino et al 17 Infantino et al 14 High signal intensity of DIA/LIA measured by densitometric quantitation Cavazzana et al 7 Bundell et al 18 Lecouffe-Desprets et al 19 No coexisting MSAs (ie, isolate antibody reactivity)…”

Dermatomyositis autoantibodies: how can we maximize utility?

“…It has more recently been confirmed that, using the Euroline, anti-Mi-2α is sensitive and specific, but anti-Mi-2β suffers from lower sensitivity and much lower specificity (154,155). It was suggested that more stringent cutoff values would improve the performance of the line assay, but this was not helpful for the performance for anti-TIF-1γ, and obviously would not ameliorate instances where sensitivity is already a problem (156,157). For example, for anti-NXP2 and anti-SAE-1, there are significant numbers of "false" positives on the Euroline (e.g., not positive on IP), while the line blot appears to have an issue with lower sensitivity in detecting anti-NXP2 antibodies (much as the case for anti-TIF-1γ) (149,(151)(152)(153).…”