Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Summary

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Abstract

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

Introduction

Antibodies labeled with small molecules are perhaps the most commonly used reagents in biology^1,2. Antibodies labeled with fluorescent dyes and biotin are extensively used in imaging, immunoassays, flow cytometry, western blots, and immunoprecipitation among other applications^3-6. Radiolabeled antibodies^3,7 find extensive use in imaging and therapy, antibodies labeled with cytotoxic drugs (ADCs) are offering new options for the treatment of cancers, and two ADCs have already been approved for therapeutic use⁸. In spite of their extensive use, the methods for labeling antibodies have remained surprisingly unchanged and typica....

Protocol

1. Antibody Capture onto High Capacity Magnetic Protein A or Magnetic Protein G Beads

1. Uniformly re-suspend magnetic beads by gentle shaking. Keep the suspension uniform when aliquoting beads.
2. Add 50 µl of bead slurry to a 1.5 ml microcentrifuge tube. Place in the magnetic stand for 10 sec. Carefully remove the storage buffer.
3. Add 250 µl of antibody binding buffer.
4. Mix and place in the magnetic stand for 10 sec. Carefully remove the binding buffer.
5. Add 1.0 ml of the sample containing 50-100 µg of antibody to the beads. Samples can be purified antibodies or antibodies in cell media.
6. Mix sample ....

Results

A schematic for labeling antibodies with small molecules using high capacity magnetic Protein A and Protein G beads is shown in Figure 1. Antibodies captured on the magnetic Protein G beads can be labeled with small molecules, for example fluorescent dyes, using either amine chemistry which labels primary amines of the lysine amino acids or using thiol chemistry which labels at the reduced thiols in the hinge region of the antibodies. Affinity between antibodies and Prote.......

Discussion

The goal of this study was to develop a method to label antibodies, present in the cell media at low concentrations, with a variety of small molecules. Such a method will allow a large number of antibodies, during the early stages of antibody discovery, to be labeled with small molecules and screened using a biologically relevant assay. One such assay is the receptor-mediated antibody internalization assay where internalization may vary between antibodies even with similar binding affinities. Hence, it is important to sc.......

Materials

Name	Company	Catalog Number	Comments
Magne Protein A Beads	Promega Corporation	G8781
Magne Protein G Beads	Promega Corporation	G7471
AlexaFluor 532-SE (Succinimidyl Ester)	Life Technologies	A20001
AlexaFluor 532-ME (Malemide)	Life Technologies	A10255
AlexaFluor 647-ME (Maleimide)	Life Technologies	A20347
Fluorescein-ME (Maleimide)	Life Technologies	F-150
Magnetic Stand	Promega	Z5332