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Multicenter Study
. 2020 Aug 10;59(1):197-207.
doi: 10.1515/cclm-2020-0981.

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

Bing Zheng  1   2 , Rodrigo A Mora  1 , Marvin J Fritzler  3 , Minoru Satoh  4 , Donald B Bloch  5 , Ignacio Garcia-De La Torre  6 , Katherine Boylan  7 , Kathryn Kohl  7 , Mark H Wener  8 , Luis E C Andrade  9   10 , Edward K L Chan  1
Affiliations
Multicenter Study

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

Bing Zheng et al. Clin Chem Lab Med. .

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.

Keywords: GW body; NuMA; autoimmunity; multiple nuclear dots; reference materials.

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Conflict of interest statement

Competing interests: MJF has received speaking honoraria from Inova Diagnostics, Werfen International (Barcelona, Spain), Amgen Canada (Mississauga, ON, Canada), and Alexion Pharmaceuticals (New Haven, CT, USA). EKLC have also received speaking honoraria from Grifols. MJF was and/or continues to be a consultant to Inova Diagnostics, Werfen International, Alexion Pharmaceuticals, and Bio-Rad (Hercules, CA, USA). MS has contract research fund from Thermo Fisher Scientific, Inc. (Tokyo, Japan). The funding organizations played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. All other authors state no conflict of interest.

Figures

Figure 1:
Figure 1:
MND-REF validation by HEp-2 IFA, double staining, 35S-K562-IP and western blotting. (A) MND-REF was serially diluted from 1:40 to 1:1280 for HEp-2 IFA. Representative images are shown for 1:40, 1:160, and 1:640 dilutions and they all presented 6–20 nuclear discrete dots corresponding to the ICAP AC-6 pattern (https://www.anapatterns.org/view_pattern.php?pattern=6). (B) Double staining of nuclear foci (arrows) identified by MND–REF (green) and rabbit anti-PML (red). (C) Left panel: IP products using 35S-methionine-labeled K562 cell extract (35S-K562-IP) of MND-REF were analyzed by 8% SDS-PAGE and autoradiography. Right panel: IP of MND-REF pre- and post-lyophilization. The proteins immunoprecipitated by MND-REF corresponded to PIC1/SUMO-1-modified PML, HK1, KLHL 7, PDK4, and protein subunit E2, E1α, and E1β of the pyruvate dehydrogenase complex (PDC) corresponding to proteins identified by IP-MS. (D) Western blotting of MND-REF using MOLT4 (left panel; 1:2,000) or HeLa (right panel; 1:5,000) whole cell lysates. Both showed reactivity to PDK4 and protein subunit E2, E1α, and E1β of PDC corresponding to proteins identified by IP-MS.
Figure 2:
Figure 2:
GWB-REF validation by HEp-2 IFA, double staining, 35S-K562-IP and western blotting. (A) GWB-REF was serially diluted from 1:40 to 1:1,280 for HEp-2 IFA. Representative images are shown for 1:40, 1:80, and 1:160 dilutions and they all presented discrete cytoplasmic dots corresponding to the ICAP AC-18 pattern (https://www.anapatterns.org/view_pattern.php?pattern=18). At 1:40 dilution, some nuclear staining is also observed. (B) Double staining of cytoplasmic foci (arrows) of GWB–REF (green) and rabbit anti-Rck/p54 (red). (C) Left panel: IP analysis of GWB–REF as described in Figure 1C. Right panel: IP comparison of GWB-REF pre- and post-lyophilization. The proteins immunoprecipitated by GWB-REF were recognized as Ge-1, topoisomerase I and SSA/Ro60 corresponding proteins identified by IP-MS. (D) Western blotting of GWB-REF using MOLT4 (left panel, 1:2,000) or HeLa (right panel, 1:5,000) whole cell lysates. Both of them showed the reactivity to Ge-1, topoisomerase I and Ro52 corresponding to proteins identified by IP-MS.
Figure 3:
Figure 3:
NuMA-REF validation by HEp-2 IFA, 35S-K562-IP, and western blotting. (A) Images of NuMA-REF by HEp-2 IFA using 1:40, 1:80, and 1:160 dilutions presenting fine speckled nuclear staining in interphase cells and bright fluorescence of spindle fibers and spindle poles characteristic of the ICAP AC-26 pattern (https://www.anapatterns.org/view_pattern.php?pattern=26). (B) Selected images of metaphase, anaphase, and telophase cells illustrating characteristic NuMA staining. (C) Left panel: IP analysis of NuMA–REF as described in Figure 1C. Right panel: 35S-K562-IP of NuMA-REF pre- and post-lyophilization. (D) Left panel: Western blotting of MOLT4 whole cell lysate with NuMA-REF diluted to 1:2,000. Right panel: Western blotting of HeLa whole cell lysate with NuMA-REF diluted to 1:5,000. Both of them showed the reactivity to NuMA.
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