Autoantibodies are present in many conditions such as cancer, autoimmune disorders, and transplant rejection. This affords an opportunity to develop diagnostic and prognostic assays, as well as to monitor disease progression and treatment effectiveness. There are several challenges, however.
“Only a fraction of patients develop autoantibodies to a given protein,” indicates Lisa Freeman-Cook, Ph.D., senior manager, R&D, Life Technologies.
“Thus, identification of multiple biomarkers in patient samples is important. We have developed functional protein microarrays (ProtoArrays®) that may be used to screen complex biological mixtures such as serum to detect autoantibodies directed against any of the 9,000 proteins on the microarray.”
The quality of the protein on the array is an important factor. “The ProtoArray contains proteins that are expressed in insect cells and purified under native conditions. These more closely recapitulate appropriate post-translational modifications and allow a more native protein conformation.”
Dr. Freeman-Cook says the utility of the ProtoArray for identifying biomarkers was demonstrated recently by investigators looking for novel autoantigens in rheumatoid arthritis, prostate cancer, and Alzheimer disease. But the next step is validation of identified biomarkers.
Validation studies typically assess candidate markers against an increasing number of independent patient samples. Thus throughput becomes important along with accurate measurements of sufficient sensitivity, dynamic range, and reproducibility to differentiate responders from nonresponders.”
Since the ProtoArray was not designed for high throughput, the company decided to employ Luminex xMAP technology.
“We developed the ProtoPlex™ Immune Response Assay featuring Luminex xMAP technology. No one before has paired these two technologies. The idea is to first utilize the ProtoArray to discover biomarkers of interest. Then, Luminex technology can be used for validation.”
The Luminex xMAP technology employs spectrally distinguishable fluorescent beads to which antigens of interest are coupled. “Cross-reactivity issues have not been a problem. As a result, we are able to accomplish higher levels of multiplexing (>80-plex) per assay with any antigen. We can process up to 500 samples per day with very low CV’s (<10%) and using only a few micoliters of patient serum.”
The company markets ProtoArray products and provides ProtoArray and ProtoPlex custom services. “We feel that combining these two technologies now not only supports biomarker discovery for a wide variety of diseases, but also allows validation with the familiar Luminex technology,” Dr. Freeman-Cook remarks.