 UW-Madison MRSEC |
Novel Methods for the Facile Construction of Single-chain Antibody Arrays |
Principal Investigator:
Eric Shusta - shusta@engr.wisc.edu
| Antibody-based arrays are often used for differential protein expression profiling (proteomics) and are playing important roles in basic biochemical discovery and health applications. These methodologies consist of surface immobilization of intact monoclonal antibodies in micron-sized spots that are subsequently used to capture proteins or analytes from a solution phase milieu. In general, construction of an antibody array is somewhat cumbersome and requires availability or creation of appropriate antibodies, antibody production and purification, and array spotting. Also, a major obstacle of traditional antibody arrays is the non-specific background generated by both the binding and non-binding regions of the intact antibodies. Protein fragments consisting of the minimal binding subunit of antibodies known as single-chain antibodies (scFvs) have excellent binding specificity and affinity for their ligands. In contrast to antibodies, scFvs lack the non-binding regions, can be selected in the company of competing antigens, and therefore have potential for higher specificity/sensitivity arrays. Because of these potential advantages, we have recently begun exploring the use of single-chain antibody fragments as a possible substitute for intact antibodies in antibody microarray applications. In order to overcome some of the current technical challenges mentioned above, we are proposing to combine scFvs generated from combinatorial yeast libraries directly with chemically compatible materials to increase the quality of antibody-based microarrays, while also dramatically simplifying their assembly. After cleaving yeast-produced scFvs from the cell exterior, we will immobilize them on functionalized The proposed combination of materials synthesis with scFv library technology has the potential to revolutionize the production and uses of antibody arrays. |
