Fast Screening of Serum Samples
The Stockholm researchers maintain that this bead-based technology offers an alternative to chip-based assays for the fast screening of large numbers of serum samples and has the flexibility of allowing different antibody arrays to be developed on demand, without the need to fabricate new arrays every time.
“We are collecting cohorts from about 25 different diseases,” Dr. Nilsson continued. “The aim is to carry out analysis on 384 samples per month, with 384 antibodies, which corresponds with roughly the number of validated antibodies emerging through HPR research.”
Researchers at the Natural and Medical Sciences Institute (NMI) at the University of Tuebingen developed an immunoassay technology, known as reverse phase protein microarray (RPPM), for applications including biomarker research, early drug discovery screening, and drug profiling. Based on planar waveguide technology developed at Zeptosens, a Bayer Technology Services company, the approach detects specific protein analytes in hundreds of samples simultaneously using well-characterized antibodies.
NMI maintains that the technology is ideal for investigations in phosphoproteomics (protein phosphorylation), a growing field of research that currently relies on technology involving the enrichment of phosphopeptides or proteins prior to mass spectrometry analysis.
“Drawbacks of the MS approach include the requirement for relatively large samples, time-consuming sample preparation, and problems with automation. In contrast, RPPM technology is significantly higher in throughput, and requires minimal sample and reagents,” explained Markus Templin, Ph.D., head of assay development at NMI. “The technology can be used for the comparative, multiparallel evaluation of several hundred samples, for applications ranging from preclinical toxicity and drug mechanism studies, to protein profiling in healthy versus diseased human tissues.
“It is, we believe, one of the most sensitive fluorescence-based approaches available, as only bound analytes at the surface of the chip generate fluorescence. Signals from unbound molecules in the bulk solution are not detected, which significantly increases the signal-to-noise ratio, allowing the detection of low-abundance proteins,” he added.
RPPM Services Offered
“RPPM assays can be carried out on denatured whole tissue or cell culture samples, and function in virtually the same way as a miniaturized dot blot. The method requires just 500 picoliters of denatured sample, and each spot generated by the arraying instrumentation is 200 microns, which contains about the same amount of material as a single cell,” Dr. Templin reported.
“The technology can detect proteins present at copy numbers down to 600–800 per cell, so even low-abundance proteins, which are increasingly believed to play critical roles in some signaling pathways, can be detected. The array format means we can put up to 320 sample spots onto a single array, six arrays on a chip, and analyze six chips in parallel.
Research at the NMI typically uses up to 250 antibodies, many of which are specific for phosphorylated proteins. At the same time, we use antibodies to detect the amount of total protein, and this allows us to calculate the relative degree of phosphorylation, which corresponds to the overall level of activation,” continued Dr. Templin.