Abstract
Objectives:Reference 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).Methods:Reference 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).Results:MND-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.Conclusions:MND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.
Highlights
Autoantibody assays are often used to assist in the evaluation of patients suspected of having a wide spectrum of autoimmune disorders
The validation of multiple nuclear dots (MND)-REF, GW bodies (GWB)-REF, and nuclear mitotic apparatus (NuMA)-REF, as appropriate reference materials was performed by seven laboratories affiliated with the Autoantibody Standardization Committee, a subcommittee of the International Union of Immunological Societies [4]
The three new reference materials reported here are intended to improve clinical assays, primarily in immunofluorescence assay (IFA), to detect these autoantibodies. These three reference materials could be valuable in clinical or research laboratories based on the validation results in our study (Table 2)
Summary
Autoantibody assays are often used to assist in the evaluation of patients suspected of having a wide spectrum of autoimmune disorders. Zheng et al.: NuMA, MND, and GWB reference materials standardization of HEp-2 IFA reporting, thirty anti-cell staining patterns (AC-0 to AC-29) have been described by the International Consensus on ANA Patterns (ICAP) (www.anapatterns.org) and their clinical relevance summarized to benefit clinicians in their daily work [2]. Sufficient quantity of plasmapheresis sample obtained from one single donor showing specific ANA patterns and/or antigen specificity is selected for further analysis and validated on different platforms in multiple expert autoantibody testing laboratories worldwide. These reference materials established by the Autoantibody Standardization Committee are distributed free of charge by Plasma Services Group (PSG, Huntingdon Valley, PA, USA; https:// www.plasmaservicesgroup.com/).
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