Establishment of international autoantibody reference standards for the detection of autoantibodies directed against PML bodies, GW bodies, and NuMA protein

Abstract

ObjectivesReference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA).MethodsReference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation–mass spectrometry (IP-MS).ResultsMND-REF stained 6–20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS.ConclusionsMND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.

Anti-dense fine speckled 70 (DFS70) autoantibodies: correlates and increasing prevalence in the United States

“…Instead, we show a P-value that indicates the statistical significance of the association between the factor as a whole and antibody status, based on an F-test from a statistical contrast. spectrometry, as described in the characterization of other autoantibody reference materials (33)(34)(35). No target antigen was consistently identified, though the data clearly showed that these sera did not recognize DFS70 in either Western blot or immunoprecipitation-mass spectrometry.…”

Application of collagen triple helix repeat containing‐1 and mitotic spindle apparatus antibody in small cell lung cancer diagnosis

“…MSA is an autoantibody mostly against nuclear mitotic apparatus protein, which is a vital product synthesized by the immune system in the anti‐tumor process. 15 Previous studies by our research group have shown that the MSA level was elevated in SCLC, making it an effective potential auxiliary tool for separating SCLC patients from NSCLC. 16 At the same time, this search yielded plasma MSA concentration in SCLC patients as 92.68 ± 41.86 pg/ ml.…”

“…The aberrant expression of those proteins can interfere with the localization of dynein on the spindle and induce spindle multi‐polarization leading to cell cycle instability and accelerating tumor progression. MSA is an autoantibody mostly against nuclear mitotic apparatus protein, which is a vital product synthesized by the immune system in the anti‐tumor process 15 . Previous studies by our research group have shown that the MSA level was elevated in SCLC, making it an effective potential auxiliary tool for separating SCLC patients from NSCLC 16 .…”

Strong Association of the Myriad Discrete Speckled Nuclear Pattern With Anti-SS-A/Ro60 Antibodies: Consensus Experience of Four International Expert Centers

“…Monoclonal anti-Ro60 IgG antibody A7 was detected in initial screening by ELISA and followed by using HEp-2 IFA. Further characterization of A7 included Western blot analysis using soluble lysate from MOLT-4 cells and a higher ratio of acrylamide monomer as cross-linker in an optimized polyacrylamide gel electrophoresis system (20), immunoprecipitation (IP) with lysate from HeLa cells metabolically labeled with [ 35 S]-methionine (21,22), and also IP using in vitro [ 35 S]-methionine-labeled translation products of Ro60 recombinant cDNA (19). IP was performed in the presence of unconjugated goat anti-mouse IgG/IgM affinity-purified IgG (Jackson ImmunoResearch Lab, West Grove, PA, USA) as a secondary linker to ensure binding of the putative monoclonal antibody to the protein G-Sepharose beads.…”