Journal: Frontiers in Immunology

Article Title: Autoantibody Landscape Revealed by Wet Protein Array: Sum of Autoantibody Levels Reflects Disease Status

doi: 10.3389/fimmu.2022.893086

Figure Lengend Snippet: Schematic of the autoantibody screening pipeline. In the first step, proteins were synthesized in vitro from the proteome-wide human cDNA library (HuPEX). Promotors (P), Enhancers (E), and FLAG-GST tags were fused to open reading frames of the expression clones by Gateway LR reaction. After polymerase chain reaction amplification and in vitro transcription, translation was performed using the wheat germ cell-free synthesis system. In the second step, we conducted autoantibody screening by treating proteome-wide wet protein arrays with pooled serum. In the third step, autoantibody quantification was performed by treating focused wet protein arrays with individual sera. ORF, open reading frame; SSc, systemic sclerosis; Pso, psoriasis; CA, cutaneous arteritis; MM, malignant melanoma; Ctrl, healthy control.

Article Snippet: Wheat germ cell-free crude solution expressing the target protein harboring FLAG-GST-tag was diluted and spotted onto GSH-coated glass-plates using a HORNET-NX multi-dispense system (Wako Pure Chemical Industries).

Techniques: Synthesized, In Vitro, cDNA Library Assay, Expressing, Clone Assay, Polymerase Chain Reaction, Amplification