Expanding assay dynamics: a combined competitive and direct assay system for the quantification of proteins in multiplexed immunoassays.
BACKGROUND
The concurrent detection and quantification of analytes that vary widely in concentration present a principal problem in multiplexed assay systems. Combining competitive and sandwich immunoassays permits coverage of a wide concentration range, and both highly abundant molecules and analytes present in low concentration can be quantified within the same assay.


METHODS
The use of different fluorescence readout channels allows the parallel use of a competitive system and a sandwich configuration. The 2 generated assay signals are combined and used to calculate the amount of analyte. The measurement range can be adjusted by varying the competitor concentration, and an extension of the assay system's dynamic range is possible.


RESULTS
We implemented the method in a planar protein microarray-based autoimmune assay to detect autoantibodies against 13 autoantigens and to measure the concentration of a highly abundant protein, total human IgG, in one assay. Our results for autoantibody detection and IgG quantification agreed with results obtained with commercially available assays. The use of 2 readout channels in the protein microarray-based system reduced spot-to-spot variation and intraassay variation.


CONCLUSIONS
By combining a direct immunoassay with a competitive system, analytes present in widely varying concentrations can be quantified within a single multiplex assay. Introducing a second readout channel for analyte quantification is an effective tool for spot-to-spot normalization and helps to lower intraassay variation.
Hartmann
Michael
M
NMI-Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.
Schrenk
Monika
M
Döttinger
Anette
A
Nagel
Sarah
S
Roeraade
Johan
J
Joos
Thomas O
TO
Templin
Markus F
MF
D001322:Autoanalysis; D001323:Autoantibodies / Q000032:analysis* / Q000276:immunology; D001324:Autoantigens / Q000276:immunology; D001327:Autoimmune Diseases / Q000276:immunology; D002232:Carbocyanines; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D040081:Protein Array Analysis; D015203:Reproducibility of Results
D001323:Autoantibodies; D001324:Autoantigens; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; C089187:cyanine dye 3; C085321:cyanine dye 5