Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements.
The ability to conveniently and rapidly profile a diverse set of proteins has valuable applications. In a step toward further enabling such a capability, we developed the use of rolling-circle amplification (RCA) to measure the relative levels of proteins from two serum samples, labeled with biotin and digoxigenin, respectively, that have been captured on antibody microarrays. Two-color RCA produced fluorescence up to 30-fold higher than direct-labeling and indirect-detection methods using antibody microarrays prepared on both polyacrylamide-based hydrogels and nitrocellulose. Replicate RCA measurements of multiple proteins from sets of 24 serum samples were highly reproducible and accurate. In addition, RCA enabled reproducible measurements of distinct expression profiles from lower-abundance proteins that were not measurable using the other detection methods. Two-color RCA on antibody microarrays should allow the convenient acquisition of expression profiles from a great diversity of proteins for a variety of applications.
Zhou
Heping
H
The Van Andel Research Institute, 333 Bostwick, Grand Rapids, MI 49503, USA.
Bouwman
Kerri
K
Schotanus
Mark
M
Verweij
Cornelius
C
Marrero
Jorge A
JA
Dillon
Deborah
D
Costa
Jose
J
Lizardi
Paul
P
Haab
Brian B
BB
D000906:Antibodies / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D001798:Blood Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D003116:Color; D004076:Digoxigenin / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008103:Liver Cirrhosis / Q000097:blood; D008113:Liver Neoplasms / Q000097:blood; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000906:Antibodies; D001798:Blood Proteins; D001710:Biotin; D004076:Digoxigenin