Multiplexed protein profiling on microarrays by rolling-circle amplification.
Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-alpha treatment. Because microarrays can accommodate approximately 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.
Schweitzer
Barry
B
Molecular Staging, Inc., Suite 701, 300 George Street, New Haven, CT 06511, USA.
Roberts
Scott
S
Grimwade
Brian
B
Shao
Weiping
W
Wang
Minjuan
M
Fu
Qin
Q
Shu
Quiping
Q
Laroche
Isabelle
I
Zhou
Zhimin
Z
Tchernev
Velizar T
VT
Christiansen
Jason
J
Velleca
Mark
M
Kingsmore
Stephen F
SF
D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D004261:DNA Replication; D004270:DNA, Circular / Q000096:biosynthesis / Q000235:genetics; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008070:Lipopolysaccharides / Q000494:pharmacology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D020543:Proteome / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000906:Antibodies; D016207:Cytokines; D004270:DNA, Circular; D008070:Lipopolysaccharides; D020543:Proteome; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha