Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies.
Isolation of antibodies to antigens that are either unstable, exist in multiple morphologies or have very limited availability can be prohibitively difficult. Here we describe a novel technique combining the capabilities of phage display antibody technology and atomic force microscopy (AFM) that is used to isolate antibody fragments that bind to a specific morphology of the target antigen, alpha-synuclein. AFM imaging allows us to both visualize the presence and morphology of the target antigen as well as to monitor the efficiency of each step in the bio-panning process. We demonstrate that phage displayed antibodies specific to the target antigen morphology can be isolated after only two rounds of selection. The target antigen, alpha-synuclein, has been correlated with the Parkinson's disease (PD). Accumulation of alpha-synuclein fibrillar aggregates into Lewy body inclusions is a hallmark feature of PD. While alpha-synuclein can form several different aggregate morphologies including oligomers, protofibrils and fibrils, the role of these morphologies in the progression of PD is not known. The successful selection of the recombinant antibody described here can have potential therapeutic value since the single-chain fragment variable (scFv) can be expressed intracellularly to control folding and toxicity of the specific protein aggregates.
Barkhordarian
Hedieh
H
Department of Chemical and Materials Engineering, Arizona State University Tempe, AZ 85287, USA.
Emadi
Sharareh
S
Schulz
Philip
P
Sierks
Michael R
MR
D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification*; D000941:Antigens; D001483:Base Sequence; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000302:isolation & purification; D018625:Microscopy, Atomic Force; D010300:Parkinson Disease / Q000276:immunology; D019151:Peptide Library; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011506:Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D051844:alpha-Synuclein / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D007141:Immunoglobulin Fc Fragments; D019151:Peptide Library; D011506:Proteins; D011994:Recombinant Proteins; D051844:alpha-Synuclein