Reliability of antinuclear matrix protein 2 antibody assays in idiopathic inflammatory myopathies is dependent on target protein properties

Abstract

A line blotting assay (LB) is currently used to detect myositis-specific autoantibodies (MSAs) in patients with idiopathic inflammatory myopathies (IIMs), because of its simplicity; however, the sensitivity and specificity of this assay is low. The aim of this study is to evaluate the accuracy of the commercial LB in detection of antinuclear matrix protein 2 (NXP2) antibody. Seventy-seven serum samples from patients with IIMs, in which anti-NXP2 antibodies were detected through immunoprecipitation and western blotting (IP-WB) using K562 cell lysate, were enrolled. All samples were assessed by LB and IP-WB using recombinant human NXP2 whole protein (rNXP2) produced by insect cells, and the positive rates of each assay were compared. Thirty-two samples (41.6%) showed falsenegativity by LB, which includes 11 samples with negative results by IP-WB using rNXP2.Relative intensities of IP-WB using cell lysate were significantly higher in the samples with positive results by both LB and IP-WB using rNXP2, compared to samples with positive by IP-WB using rNXP2 but negative by LB. Three of 11 samples with negative results by both LB and IP-WB using rNXP2 revealed high antibody titers. Further, differences in post-transcriptional SUMOylation were observed between recombinant and natural NXP2 proteins. In conclusion, the LB showed low sensitivity for detection of anti-NXP2 antibody, an effect exacerbated at low titers of anti-NXP2 antibodies. Moreover, there

Severe gastrointestinal involvements in patients with adult dermatomyositis with anti-NXP2 antibody

“…However, immunoprecipitation (IP) was a more sensitive method in detection of anti-NXP2 antibodies. 29 Therefore, the estimation of incident rate may be improved by IP method.…”

Update on autoantibodies and related biomarkers in autoimmune inflammatory myopathies

“…Since then, some of these assays are being replaced by newer and more robust technologies such as dot immunoblot assays (DIA) and line immunoassays (LIA), addressable laser bead immunoassays (ALBIA), particle-based multianalyte technology (PMAT), mass spectrometry and immunoprecipitation mass spectrometry (IP-MS) [23][24][25][26]. However, some of these novel assays that had gained favor as high throughput, multiplexed and relatively economical technologies, are faced by technical and performance challenges [25,[27][28][29][30][31][32][33]. A narrative review the methods (IP, ELISA, LIA, ALBIA, DIA) used to detect AIM-A and AIM-S autoantibodies in AIM has recently been published [34 && ].…”

Update on dermatomyositis

“…This is likely because differences in posttranscriptional modification (i.e. SUMOylation) in natural and recombinant proteins affect the antibody recognition [23 ▪ ]. The other limitations include environmental temperature affecting line blot performance and causing discordant results in different laboratories [24] and serum with low-antibody titers, especially anti-TIF1-γ and anti-NXP-2, may not be detected by line blot [21 ▪ ,23 ▪ ].…”

“…SUMOylation) in natural and recombinant proteins affect the antibody recognition [23 ▪ ]. The other limitations include environmental temperature affecting line blot performance and causing discordant results in different laboratories [24] and serum with low-antibody titers, especially anti-TIF1-γ and anti-NXP-2, may not be detected by line blot [21 ▪ ,23 ▪ ]. Considering these limitations and pitfalls, standardization and harmonization for the antibody detection methods is essential and requires multicenter study collaborations.…”