Double-chip protein arrays: force-based multiplex sandwich immunoassays with increased specificity.
Protein assays provide direct access to biologically and pharmacologically relevant information. To obtain a maximum of information from the very smallest amounts of complex biological samples, highly multiplexed protein assays are needed. However, at present, cross-reactions of binding reagents restrict the use of such assays to selected cases and severely limit the potential for up-scaling the technology. Here we describe a double-chip format, which can effectively overcome this specificity problem for sandwich immunoassays. This format consists of a capture array and a reference array with fluorescent labeled detection antibodies coupled to the reference array via DNA duplexes. This format allows for the local application of the labeled detection antibodies onto their corresponding specific spots on the capture array. Here we show that this double-chip format allows for the use of cross-reactive antibodies without generating false positive signals, and an assay for the parallel detection of seven different cytokines was set up. Even without further optimization, the dynamic range and the limit of detection for interleukin 8 were found to be comparable to those obtained with other types of multiplexed sandwich immunoassays.
Blank
Kerstin
K
nanotype GmbH, Lochhamer Schlag 12, 82166 Gräfelfing, Germany.
Lankenau
Andreas
A
Mai
Thao
T
Schiffmann
Susanne
S
Gilbert
Ilka
I
Hirler
Siegfried
S
Albrecht
Christian
C
Benoit
Martin
M
Gaub
Hermann E
HE
Clausen-Schaumann
Hauke
H
D000818:Animals; D000906:Antibodies / Q000032:analysis; D016207:Cytokines / Q000032:analysis; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods; D051379:Mice; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013499:Surface Properties
D000906:Antibodies; D016207:Cytokines