Development of a "reverse capture" autoantibody microarray for studies of antigen-autoantibody profiling.
Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.
Qin
Shuzhen
S
Molecular Urology Laboratory, Division of Urology, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA.
Qiu
Weiliang
W
Ehrlich
Joshua R
JR
Ferdinand
Angeline S
AS
Richie
Jerome P
JP
O'leary
Michael P
MP
Lee
Mei-Ling Ting
ML
Liu
Brian C-S
BC
D000368:Aged; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000097:blood* / Q000276:immunology; D001323:Autoantibodies / Q000097:blood*; D014408:Biomarkers, Tumor / Q000097:blood*; D015153:Blotting, Western; D002232:Carbocyanines; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D008875:Middle Aged; D011470:Prostatic Hyperplasia / Q000276:immunology; D011471:Prostatic Neoplasms / Q000276:immunology; D040081:Protein Array Analysis; D044967:Serum
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D001323:Autoantibodies; D014408:Biomarkers, Tumor; D002232:Carbocyanines; D005456:Fluorescent Dyes; C089187:cyanine dye 3