Journal: bioRxiv
Article Title: Symmetric Dimethylarginine Modification Drives Shared and Divergent Antigen Reactivity in Lupus
doi: 10.64898/2026.09.22.753525
Figure Lengend Snippet: (A) Sera from MRL lpr mice (n=12, bled weekly from 8 to 13 weeks of age) were analyzed by ELISA using synthetic 10×RG peptides: native sequence (GRGRGRGRGRGRGRGRGRGR) vs. SDMA-modified (lowercase ‘r’ denotes SDMA; GrGrGrGRGRGrGrGrGRGR). All 12 mice developed anti SDMA antibody titers above the corresponding native controls, and the majority (9/12) showed a gradual increase in anti-SDMA titers over time. (B) Immunoblotting of HEp-2 lysates revealed progressive changes in band patterns from 10 weeks onward. To identify SDMA specific reactive proteins, cells were pretreated with the methyltransferase inhibitor EPZ015666 (EPZ) before lysis. A prominent ∼15 kDa band (arrowhead) was markedly reduced by EPZ pretreatment, supporting its recognition by anti SDMA antibodies. (C) Sera from three 12 week old MRL lpr littermates (Lpr 1, 2, 3) each showed distinct band patterns against the cell lysates. EPZ pretreatment of the cells reduced the intensity of several bands in all three mice. (D) Workflow for isolating hybridoma clones from individual plasma cells to obtain monoclonal antibodies (mAbs) specific for SDMA. MRL lpr mice were not immunized with any antigen; thus, the resulting mAbs represent naturally arising autoantibodies. (E) Two antibody clones were tested against the native versus SDMA peptide array shown in Figurè3. Clone P4A2F4 reacted extensively with most SDMA containing peptides, whereas clone P2C1C10 showed no reactivity. (F) The two mAbs produced different band patterns on cell lysates. P4A2F4 recognized bands that were reduced in EPZ pretreated lysates, consistent with SDMA specificity. (G) After implantation of the SDMA-reactive hybridoma clone into the peritoneal cavity of healthy BALB/c mice, the kidneys showed extensive IgG deposition, reminiscent of human lupus nephritis.
Article Snippet: High-binding ELISA plates (Greiner Bio-One, 655081) were coated with 250 ng/well of streptavidin (Leinco Technologies, S203) overnight at 4°C.
Techniques: Enzyme-linked Immunosorbent Assay, Sequencing, Modification, Western Blot, Lysis, Clone Assay, Clinical Proteomics, Bioprocessing, Autoantibodies, Peptide Microarray, Produced