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. 2009 Feb 16;206(2):449–462. doi: 10.1084/jem.20081862

Figure 1.

Figure 1.

ACC mAb antibody specificity in vitro and in vivo. (a) ACC mAb specificity in ELISA. Several arginine-containing peptides and their citrulline analogues have been tested to characterize the citrulline specificity of our generated antibodies. Some of the epitopes were accessible in the triple helical form of the citrulline-containing C1III epitope (THPCII-Cit) for some of the antibodies (ACC1–3 and ACC5; A and B). Nevertheless, this epitope seems not to be accessible for ACC4. Some of the antibodies cross react to selected citrullinated peptides even with a noncollageneous backbone. ACC2, ACC3, and ACC5 bind not only to CII but also to the citrullinated derivative of fibrinogen (C and D). Furthermore, ACC4 and ACC5 bind stronger to the citrullinated form of cyclic filaggrin (cyc-Cit) than to its arginine-containing form (cyc-Arg; E and F). Enzymatic deimination of CII with PAD4 generates neoepitopes, which are recognized by ACC2–4 (G–I). C1III peptides containing an additional biotinylated lysine have been synthesized, which in turn bind to NeutrAvidin-precoated ELISA plates (J–M). This setup allows free accessibility of the different antibodies specific for the citrulline-modified immunodominant CII epitopes. All of the assays were done in duplicate. (b) Staining of arthritic joints. Citrulline-specific antibodies bind to arthritic cartilage. Joint sections (10 mm) from arthritic BALB/c (A, C, and D), naive (BALB/c × B10.Q) F1 (B), or BALB/c (E) mice are shown. Results shown are representative histological pictures of arthritic (n = 4) and control (n = 3) mice used in the staining of joints with anticitrulline antibodies. Arthritis was induced in 4–6-mo-old naive male BALB/c mice (n = 36) by injecting 9 mg of an arthritogenic anti-CII mAb cocktail containing antibodies M2139 (binding to the J1 epitope) and CIIC1 (binding to the C1I epitope). The arthritis induction experiment was performed four times independently with 100% incidence and a mean maximum arthritis score of 32.9 ± 3. The four mice that were used for histology had arthritis scores of 59, 60, 47, and 50. Paw samples were taken on day 11 after antibody transfer (5 d after LPS injection). Sections were treated with no antibodies (A), ACC1 (B), or ACC4 (C–E) mAbs. Magnification, ×10.