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 typically involve multiple reactions and desalting steps^9-12. Solution methods work very well in cases where only a few antibodies need to be labeled and are available in highly purified form at high concentration and in sufficient volumes. However, for newer applications like ADCs, there is a need to label antibodies at the early hybridoma stage so that they can be screened for biologically relevant properties, for example, receptor-mediated antibody internalization^13-16. At the hybridoma stage, sample volumes are limited, antibodies are expressed at low concentrations and a number of samples are large, hence solution based labeling methods are not suitable.

To simplify and improve the throughput of the traditional antibody labeling methods, a few alternative approaches have been proposed^17,18. One approach is to use non-magnetic Protein A affinity beads packed in small columns to capture antibodies followed by the labeling reaction and elution of labeled and purified protein. This method can be used to label antibodies directly from cell media, however, the use of columns can be laborious. A magnetic bead based method has recently been reported¹⁹ that eliminates the use of columns and improves throughput but due to the limited antibody binding capacity of the beads, only nanogram to low microgram quantities of the antibodies could be labeled.

We recently developed and used high capacity magnetic Protein A and Protein G beads (>20 mg of Human IgG/ml of settled beads) to label antibodies present in cell media with small molecules²⁰. The high capacity of the beads allows tens to hundreds of micrograms of antibody to be labeled conveniently and the rapid magnetic response of the beads simplifies handling and processing of a large number of samples in parallel. Using fluorescent dyes as surrogates for small molecules, we show that the method is compatible with amine and thiol labeling chemistry and offers high recoveries of labeled and very pure antibodies.

This protocol and the accompanying video describe on-bead labeling of mouse antibodies present in the cell media using Magnetic Protein A and Protein G beads. The protocol is divided into four sections: Section 1 describes the capture of antibodies onto the bead from biological samples. Following capture, the labeling of antibodies with fluorescent dye using amine chemistry or using thiol chemistry is described in sections 2 and section 3, respectively. Finally, section 4 describes the method for the calculations of the antibody concentration and the dye to antibody ratio.

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 for 60 min at RT using a vortex mixer or end-over-end mixer.
7. Place tube in the magnetic stand for 10 sec and remove the supernatant.
8. Add 250 µl of antibody binding/washing buffer and mix. Place in the magnetic stand for 10 sec and remove binding/washing buffer. Repeat this step for a total of two washes.
9. Proceed to Section 2 to label antibodies using amine chemistry or to Section 3 to label antibodies using thiol chemistry.

2. Antibody Labeling Using Amine Chemistry

1. Conjugate Antibody
   1. Add 100 µl of amine conjugation buffer to the beads.
   2. Dissolve the amine-reactive fluorescent dyes (AlexaFluor 532-SE) at 10 mg/ml by adding 100 µl of Dimethyl sulfoxide (DMSO) to 1.0 mg of dye. Mix by vortexing. Make this solution just before use.
   3. Add 2.5 µl of amine-reactive dye for 100 µg of antibody.
      Note: Typically a 5-20 molar excess of reactive dye is recommended. However, amount of reactive dye added to the reaction needs to be empirically optimized depending on desired dye to antibody ratio and intrinsic antibody properties.
   4. Mix sample for 60 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.
   5. Place tube in the magnetic stand for 10 sec and remove the supernatant.
   6. Add 250 µl of antibody binding/washing buffer and mix. Place in the magnetic stand for 10 sec. Remove and discard binding/washing buffer. Repeat this step for a total of two washes.
2. Antibody Recovery
   1. Add 50 µl of elution buffer to the beads.
   2. Mix for 5 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.
   3. Place tube in the magnetic stand for 10 sec. Remove eluted sample and transfer to a new micro-centrifuge tube containing 5 µl of neutralization buffer.
   4. Repeat the process one more time and pool the eluted samples.
   5. Quantitate the antibody concentration and dye-to-antibody ratio as described in Section 4.

3. Antibody Labeling Using Thiol Chemistry

1. Antibody Reduction
   1. Add 250 µl of thiol conjugation buffer and mix. Place the tube in the magnetic stand for 10 sec. Remove and discard the buffer. Repeat this step twice.
   2. Add 100 µl of thiol conjugation buffer.
   3. Add Dithiothreitol (DTT) to a final concentration of 2.5 mM.
   4. Mix sample for 60 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.
   5. Place the tube in the magnetic stand for 10 sec and discard the buffer.
   6. Add 250 µl of thiol conjugation buffer and mix. Place the tube in the magnetic stand for 10 sec. Remove and discard the buffer. Repeat this step for a total of two washes.
   7. Add 100 µl of thiol conjugation buffer.
2. Conjugate Antibody
   1. Dissolve the thiol-reactive dye at 10 mg/ml by adding 100 µl of DMSO to 1.0 mg of dye. Mix by vortexing. Make this solution just before use.
   2. Add 2.5 µl of thiol-reactive dye for 100 µg of antibody.
      Note: Typically a 5-20 molar excess of reactive dye is recommended. However, amount of reactive dye added to the reaction needs to be empirically optimized depending on desired dye to antibody ratio and intrinsic antibody properties.
   3. Mix sample for 60 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.
   4. Place the tube in the magnetic stand for 10 sec and remove supernatant.
   5. Add 250 µl of thiol conjugation buffer and mix. Place in the magnetic stand for 10 sec and remove the buffer. Repeat this step for a total of two washes.
3. Elute Antibody
   1. Add 50 µl of elution buffer to the beads.
   2. Mix for 5 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.
   3. Place tube in the magnetic stand for 10 sec. Remove eluted sample and transfer to a new micro-centrifuge tube containing 5 µl of neutralization buffer.
   4. Repeat the process one more time and pool the eluted samples.
   5. Quantitate the antibody concentration and dye-to-antibody ratio as described in Section 4.

4. Calculate Dye-to-Antibody Ratio

1. Measure the absorbance of the antibody-dye conjugate at 280 nm (A280) and at the λmax for the dye (Amax).
2. Calculate the antibody concentration:
   Antibody Concentration (mg/ml) = A280 - (A_max × CF) / 1.4
   where CF = Correction factor of the dye (provided by the manufacturer).
3. Calculate the dye-to-antibody ratio:
   Dye-to-Antibody Ratio (DAR) = (A_max × 150,000)/Ab Concentration (mg/ml) × ε_dye
   where, ε_dye = the extinction coefficient of the dye at its absorbance maximum and molecular weight of antibody = 150,000 Da.

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