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

Summary

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved 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.

Introduction

The presence of protein variants has been implicated as a factor in the progression of many diseases including neurodegenerative diseases such as Alzheimer’s, Parkinson’s, ALS and Frontotemporal Dementia (FTD)^{1,2,3,4,5,6,7,8,9,10,11}. Oligomeric forms of the proteins beta-amyloid and alpha-synuclein are thought to be the toxic species responsible for Alzheimer’s and Parkinson’s, respectively^2,3,4,5. Aggregates of the TAR DNA-binding protein 43 (TDP-43) have been linked to ALS and FTD^12,13,14. Therefore reagents such as antibodies that can selectively target the different protein variants can be powerful tools to ....

Protocol

1. Phage Production

1. Perform all phage production and biopanning processes in a biosafety cabinet. Produce phages particles from the different libraries (Tomlinson I and J libraries and Sheets library²¹) for the biopanning process using the manufacturer’s instructions (http://www.lifesciences.sourcebioscience.com/media/143421/tomlinsonij.pdf).
   NOTE: We use multiple libraries in our panning process to increase the diversity of the antibodies available.
2. Briefly, culture bacteria stocks of the three libraries in 200 ml of 2xYT containing 1% glucose and 100 μg/ml of ampicillin on a shaker at 37 °C until OD₆₀₀

Results

In Figure 1, the schematic demonstrates the negative panning process by which we removed phage binding off-target antigens from our library using immunotubes. We initially started with BSA since this is a common blocking agent and any phage that would react nonspecifically with this target would be problematic in future immunoassays. Next, we removed binders to aggregated alpha-synuclein to eliminate phage that are reactive with generic structures of aggregated proteins (i.e., an antibody that w.......

Discussion

Protein variants have been shown to be involved in the progression of many neurodegenerative diseases such as Alzheimer’s, Parkinson’s, ALS and FTD^{1,2,3,4,5,6,7,8,9,10,11}. Isolation of antibodies that can selectively recognize these different protein variant targets can be effective reagents to study, diagnose and potentially treat such ailments. To generate such variant specific antibodies we have developed a novel biopanning process that utilizes atomic force microscopy to monitor the progress .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tomlinson I and J Libraries	MRC (Cambridge, England)
Sheets Library	MRC (Cambridge, England)
2xYT	BD Sciences	244020
Glucose	Amresco	0188-2.5KG
Ampicillin	Amresco	0339-25G	Irritant
KM13 Helper Phage	MRC (Cambridge, England)
Kanamycin	OmniPur	5880	Irritant
Polyethylene Glycol 8000	OmniPur	6510	Irritant
Sodium Chloride	Macron	7647-14-5
Sodium Phosphate Dibasic	Amresco	0404-1KG	Irritant
Potassium Chloride	EMD	PX1405-1	Irritant
Potassium Phosphate Monobasic	Amresco	0781-500G	Irritant
TG1 Cells	MRC (Cambridge, England)
Luria-Bertani Agar	EMD	1.10283.0500
Bovine Serum Albumin	Amresco	0332-100G
STEN buffer	Crystalgen Inc.	33429775
Immunotubes	Thermo Scientific	470319
Mica	Spruce Pine Mica	24365
Tween 20	EMD
Trypsin	Sigma	T-0303	Irritant
Triethylamine	Sigma	T-0886	Flammable
Glycerol	Amresco	0854-1L	Irritant
DNA Plasmid Prep Kit	qiagen	27106	Irritant
Non-Fat Milk Powder	Carnation
96-Well High Binding ELISA Plate	Costar	3590
Anti-M13 HRP	GE Healthcare Life Sciences	27-9421-01
ELISA Femto Chemiluminescence Substrate Kit	Thermo Scientific	37074
Anti-TDP 43 Polyclonal Antibody	ProteinTech	10782-2-AP
A/G Agarose Beads	Santa Cruz Biotechnology	sc-2003
HB 2151 Cells	MRC (Cambridge, England)
Isopropylthiogalactoside	Teknova	13325
9e10 HRP	Santa Cruz Biotechnology	sc-40
Nitrocellulose Membrane	Biorad	162-0115	Flammable
Centrifuge	Thermo Scientific	Sorvall RC 6+
Nanoscope IIIa Atomic Force Microscope	Veeco
AFM Probes	VistaProbes	T300R-10