Development of a protein microarray for profiling circulating autoantibodies in human diseases.

Abstract

To develop a robust microarray platform to detect thousands of serological autoantibodies (AAbs) simultaneously in different diseases. An AAbMap microarray was prepared by printing a total of 4032 purified His-tagged human proteins and peptide probes on a chemically-modified slide. The sensitivity, dynamic range, and the inter- and intra-array reproducibility of the AAb microarray were then systematically tested and optimized. Finally, the large-scale profiling of AAbs in the serum of patients with different human diseases using the AAbMap microarray was demonstrated. The dynamic range of antibody (Ab) detection was 2 to 3 orders of magnitude with the lowest limit of detection (LOD) of 68 pg/mL. The intra-array (r) correlation of duplicate spots was 1.00, whereas the inter-array correlations between different arrays and batches were 0.99 and 0.97 to 0.98, respectively. Notably, 132, 266, 171, and 84 AAbs were detected in pooled serum from healthy controls (HCs) or patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or lung cancer (LC), respectively. These AAbs included antibodies that target well-known disease biomarkers, such as anti-cyclic citrullinated peptide, anti-ribonucleoprotein, and anti-nucleosome. We developed a microarray platform to measure thousands of serological AAbs simultaneously with high sensitivity and reproducibility. The array can help study autoimmunity and complement genomics, proteomics, and metabolomics data for systematic investigations of human diseases.