Protein Arrays for Biomarker Detection

Antibodies to tumor antigens have advantages over other serum proteins as potential cancer biomarkers as they are stable, highly specific, easily purified from serum, and are readily detected with well-validated secondary reagents. The technologic development of protein microarrays offers an emerging platform to present tumor antigens to screen for immune responses.  In comparison to traditional ELISAs, protein microarrays are capable of presenting and assessing hundreds of tumor antigens simultaneously.  The responses are rapidly identified because the address of each protein is known in advance and there are no representation issues; all proteins, even rare ones, are represented equally. The proteins are arrayed on a single microscope slide or on bead arrays requiring only a few microliters of serum per assay. We have developed novel assays for antibody detection using a protein microarray technology, termed Nucleic Acid Protein Programmable Array (NAPPA).

The NAPPA approach entails programming cell-free protein expression extracts with cDNAs to express the proteins at the time of the assay without the need for advanced purification.  This strategy replaces purified proteins with cDNAs encoding the target proteins at each feature of the array.  The proteins are then transcribed and translated by a cell-free system and immobilized in situ using epitope tags fused to the proteins.  This approach eliminates the need to express and purify proteins separately and produces proteins “just-in-time” for the assay, abrogating concerns about protein stability during storage.  This chemistry also has the advantage that mammalian proteins can be expressed in a mammalian milieu (reticulocyte lysate) to increase the efficiency of expression and to encourage natural folding of the proteins.

We have used these microarrays to express 5000 human tumor antigens to screen sera from patients with breast and ovarian cancers.  We have identified panels of potential autoantibody biomarkers for these cancers, including antibodies to p53 protein.  These biomarkers are now in validation studies through the NCI/Early Detection Research Network.