Two-dimensional electrophresis and mass spectrometry identification of proteins bound by a murine monoclonal anti-cardiolipin antibody: A powerful technique to characterize the cross-reactivity of a single autoantibody

Thebault, Sabine; Gilbert, Danièle; Machour, Nadine; Marvin, Laure; Lange, Catherine; Tron, François; Charlionet, Roland

doi:10.1002/1522-2683(20000701)21:12<2531::aid-elps2531>3.3.co;2-5

Cited by 4 publications

(9 citation statements)

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“…Similarly, antinucleophosmin autoantibodies were previously detected in SLE patients, and were shown to be constantly associated with the presence of anti-CL Abs (23). It is worth noting that two monoclonal autoantibodies, one derived from the PBL of an SLE patient, and the other from male WB splenocytes, and both selected based on their capacities to react with anionic phospholipids, also bound to vimentin (24) and nucleophosmin (19), respectively. These observations, and the high frequency of anti-vimentin and anti-FIGURE 7.…”

Section: Discussionmentioning

confidence: 99%

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Orderly Pattern of Development of the Autoantibody Response in (New Zealand White × BXSB)F1 Lupus Mice: Characterization of Target Antigens and Antigen Spreading by Two-Dimensional Gel Electrophoresis and Mass Spectrometry

Thebault

Gilbert

Hubert

et al. 2002

The Journal of Immunology

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Immunoblots of a two-dimensional PAGE-separated HL-60 cell proteomic map and mass spectrometry were combined to characterize proteins targeted by autoantibodies produced by male (New Zealand White × BXSB)F1 (WB) mice that develop lupus and anti-phospholipid syndrome. Analysis of sera sequentially obtained from seven individual mice at different ages showed that six proteins, vimentin, heat shock protein 60, UV excision-repair protein RAD23, α-enolase, heterogeneous nuclear ribonucleoprotein L, and nucleophosmin, were the targets of the B cell autoimmune response, and that autoantibodies to them were synthesized sequentially in an orderly pattern that recurred in all the male WB mice analyzed: anti-vimentin first and anti-nucleophosmin last, with anti-RAD23 and anti-heat shock protein 60, then anti-α-enolase and anti-heterogeneous nuclear ribonucleoprotein L Abs occuring concomitantly. Anti-vimentin reactivity always appeared before anti-cardiolipin and anti-DNA Abs, suggesting that vimentin is the immunogen initiating the autoimmune process. The pattern of HL-60 proteins recognized by female WB sera differed from that of male sera, indicating that the Y chromosome-linked autoimmune acceleration gene is not an accelerator but a strong modifier of the autoimmune response. Thus, 1) combining two-dimensional PAGE and mass spectrometry constitutes a powerful tool to identify the set of Ags bound by autoantibodies present in a single serum and the whole autoantibody pattern of an autoimmune disease; 2) the diversification of the autoimmune response in male WB mice occurs in a predetermined pattern consistent with Ag spreading, and thus provides a useful model to further our understanding of the development of the autoantibody response in lupus.

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Section: Discussionmentioning

confidence: 99%

“…All reagents and instruments used have been described in detail elsewhere (19). Protein sample preparation.…”

Section: D-page Map Constructionmentioning

confidence: 99%

Orderly Pattern of Development of the Autoantibody Response in (New Zealand White × BXSB)F1 Lupus Mice: Characterization of Target Antigens and Antigen Spreading by Two-Dimensional Gel Electrophoresis and Mass Spectrometry

Thebault

Gilbert

Hubert

et al. 2002

The Journal of Immunology

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“…The immunofluorescence pattern of sera from WB mice and patients with SLE was determined on Hep-2 cells as described previously [ 10 ]. Inhibition experiments were performed by preincubating an anti-NPM mouse mAb (Invitrogen immunodetection, San Francisco, CA, USA), mouse and patient sera for 1 hour with 20 μg of recombinant NPM.…”

Section: Methodsmentioning

confidence: 99%

“…We previously observed that WB mouse-derived monoclonal antibodies (mAbs) selected for their capacities to react with CL in the presence of FCS also reacted with nuclear antigens, as shown by their nucleolar immunofluorescence-labelling pattern on HEp-2 cells [ 10 ]. One of these mAbs, 4B7, reacted with nucleophosmin (NPM; also known as B23), a nucleolar protein involved in the assembly and transport of ribosomes [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

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Lartigue

Drouot

Jouen³

et al. 2005

Arthritis Res Ther

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We showed previously that nucleophosmin (NPM), a nucleolar phosphoprotein, is recognized by sera from (NZW × BXSB)F 1 (WB) mice, a model of systemic lupus erythematosus (SLE) and anti-phospholipid syndrome. In the present study we analysed the prevalence and kinetics of anti-NPM autoantibodies in WB mice by a solid-phase ELISA with recombinant human (rh) NPM as the antigen and showed that most male WB mouse sera had anti-NPM antibodies that were responsible for their indirect immunofluorescence staining pattern on Hep-2 cells. Anti-NPM antibodies were significantly associated with anti-cardiolipin (aCL) antibodies. This antibody profile mirrored that observed in certain human SLE sera because anti-NPM antibodies were detected in 28% of the sera from patients with SLE and were similarly associated with aCL antibodies. The demonstration that rhNPM bound to cardiolipin (CL) in vitro and increased the CL-binding activity of a WB-derived aCL monoclonal antibody indicates that NPM can interact with CL to form SLE-related immunogenic particles that might be responsible for the concomitant production of anti-NPM and aCL antibodies.

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“…Cela constitue le principal avantage et l'originalité de cette technologie, largement utilisée pour l'exploration du répertoire des auto-anticorps dans diverses pathologies et pour l'analyse des processus physiopathologiques à l'oeuvre dans les maladies auto-immunes [19][20][21][22][23]. Son principal inconvénient réside dans la lourdeur de sa mise oeuvre et de sa standardisation.…”

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