Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Abstract

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson's disease and beta-amyloid and tau in Alzheimer's disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases. Video LinkThe video component of this article can be found at

TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis

“…S2) and had stronger binding to ALS compared to both FTD and control samples (Additional file 3: Fig. S3) confirming the efficiency of our previously described biopanning protocol for generating reagents that bind disease specific protein variants [32]. …”

“…Phage expressing the different anti-TDP-43 scFvs were previously selected for preferential binding to ALS brain tissue samples relative to both control and FTD cases as described [32]. We characterized the binding of 23 different phage-displayed scFvs toward ALS, FTD and healthy human brain tissue samples from the motor cortex using a simple indirect phage ELISA (Additional file 1: Fig.…”

“…This protocol was also utilized to generate scFvs against variants of TDP-43 present in human ALS cases (Stage 1A from Additional file 1: Fig. S1) [32]. We immunoprecipitated TDP-43 proteins from the homogenized motor cortex of ALS and control cases using a commercially available polyclonal anti-TDP-43 antibody as previously described [32].…”

“…S1) [32]. We immunoprecipitated TDP-43 proteins from the homogenized motor cortex of ALS and control cases using a commercially available polyclonal anti-TDP-43 antibody as previously described [32]. Since TDP-43 pathology also exists in around 45% of frontotemporal dementia (FTD) cases [5, 33–37], we also included multiple rounds of negative panning against TDP-43 variants immunoprecipitated from the motor cortex of FTD cases.…”

“…After biopanning, we identified 23 different complete scFv sequences that all preferentially bound ALS tissue over both FTD and healthy samples using indirect phage ELISAs (Stage 2 from Additional file 1: Fig. S1) [32]. …”