High-throughput analysis of kinetic ELISA-microarray data

Protein microarrays are widely used in proteomics, systems biology or biomarker discovery for sensitive high-throughput analysis of larger number of proteins in a single biological or clinical sample. ELISA technology applied to protein microarrays allows for enhancement of signal specificity and due to enzymatic amplification also promises higher sensitivity. However, the specific properties of enzymatic signal amplification can limit the quantification range of the assay due to non-linear signal development upon increase of analyte concentration. We extended the detection range using a kinetic colorimetric detection protocol, which in comparison to end-point measurements follows the amplified signal over time. For the analysis of the raw data we developed a computational approach implemented in R, which can handle three-dimensional data obtained by on-line recording of the enzymatic signal and significantly enhances the precision and quantification. With this approach we detect protein concentration over a range of 3-4 orders of magnitude. We have successfully utilized the quantitative kinetic detection with ArrayTubeTM and ArrayStripTM micorarrays (Alere Technology GmbH, Jena, Germany) designed for the analysis of phosphorylated proteins of key signal transduction pathways. We are convinced that our quantification method can be used for all ELISA systems, and should be of interest for other bioanalytical applications, in which kinetic amplification of the detection signal can be followed over time. Due to the high sensitivity and the broad quantification range, kinetic ELISA microarrays can be a promising instrument to study signal transduction, protein modifications, and provide accurate quantitative data for modelling of signalling pathways.