Beta Amyloid~Isolation and characterization of antibody fragmentsselective for specific protein morphologies from nanogram antigensamples.

We developed atomic force microscope (AFM) based protocols that enable isolation and characterization of antibody based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single chain antibody fragments (scFvs) that specifically recognize anoligomeric amyloid-beta (Aβ) species correlated with Alzheimer's disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We employedseveral subtractive panning steps to remove all phagebinding non-desired antigens and then employed a single positive panning step using minimal antigen. We also used AFM to characterize the specificity of the isolated clones,again using minimal material, selecting the C6 scFv based on expression levels. We show that C6selectively bindscell and brain derived oligomeric Aβ. The protocols described are readily adapted to isolating antibody based reagents against other antigenic targets with limited availability. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.