1. Antibody Capture onto High Capacity Magnetic Protein A or Magnetic Protein G Beads
<ol>
	<li>Uniformly re-suspend magnetic beads by gentle shaking. Keep the suspension uniform when aliquoting beads.</li>
	<li>Add 50 &#181;l of bead slurry to a 1.5 ml microcentrifuge tube. Place in the magnetic stand for 10 sec. Carefully remove the storage buffer.</li>
	<li>Add 250 &#181;l of antibody binding buffer.</li>
	<li>Mix and place in the magnetic stand for 10 sec. Carefully remove the binding buffer.</li>
	<li>Add 1.0 ml o...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+modified+antibodies+as+diagnostic+imaging+agents.">Chemically modified antibodies as diagnostic imaging agents.</a> Curr Opin Chem Biol. 14 (6), 803-809 (2010).</li><li>Cunningham, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+flow+cytometry+and+fluorescent+probes+for+flow+cytometry.">Overview of flow cytometry and fluorescent probes for flow cytometry.</a> Methods Mol Biol. 588, 319-326 (2010).</li><li>Eaton, S. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent-labeled+antibodies:+Balancing+functionality+and+degree+of+labeling.">Fluorescent-labeled antibodies: Balancing functionality and degree of labeling.</a> Anal Biochem. 402 (2), 146-150 (2010).</li><li>Isa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+system+to+identify+small+molecules+that+induce+internalization+and+degradation+of+HER2.">High-throughput screening system to identify small molecules that induce internalization and degradation of HER2.</a> ACS Chem Biol. 9 (10), 2237-2241 (2014).</li><li>Liao-Chan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+antibody+internalization+with+novel+monoclonal+antibodies+against+Alexa+fluorophores.">Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.</a> PLoS One. 10 (4), e0124708 (2015).</li><li>Riedl, T., van Boxtel, E., Bosch, M., Parren, P. W., Gerritsen, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screening+for+Internalizing+Antibodies+by+Homogeneous+Fluorescence+Imaging+of+a+pH-Activated+Probe.">High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe.</a> J Biomol Screen. 21 (1), 12-23 (2016).</li><li>Nath, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+plate+based+antibody+internalization+assay+using+pH+sensor+fluorescent+dye.">Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.</a> J Immunol Methods. 431, 11-21 (2016).</li><li>Lundberg, E., Sundberg, M., Graslund, T., Uhlen, M., Svahn, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+reproducible+fluorescent+labeling+of+small+amounts+of+antibodies+on+solid+phase.">A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase.</a> J Immunol Methods. 322 (1-2), 40-49 (2007).</li><li>Strachan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+biotinylation+of+antibodies.">Solid-phase biotinylation of antibodies.</a> J Mol Recognit. 17 (3), 268-276 (2004).</li><li>Dezfouli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+bead+assisted+labeling+of+antibodies+at+nanogram+scale.">Magnetic bead assisted labeling of antibodies at nanogram scale.</a> Proteomics. 14 (1), 14-18 (2014).</li><li>Nath, N., Godat, B., Benink, H., Urh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-bead+antibody-small+molecule+conjugation+using+high-capacity+magnetic+beads.">On-bead antibody-small molecule conjugation using high-capacity magnetic beads.</a> J Immunol Methods. 426, 95-103 (2015).</li><li>Lund, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+variation+in+binding+of+Protein+A+and+Protein+G+to+immunoglobulin+type+G+by+isothermal+titration+calorimetry.">Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry.</a> J Mol Recognit. 24 (6), 945-952 (2011).</li><li>Safarik, I., Safarikova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+techniques+for+the+isolation+and+purification+of+proteins+and+peptides.">Magnetic techniques for the isolation and purification of proteins and peptides.</a> Biomagn Res Technol. 2 (1), 7 (2004).</li><li>Flygare, J. A., Pillow, T. 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Authors are employees of Promega Corporation.

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...

Antibodies labeled with small molecules are perhaps the most commonly used reagents in biology1,2. Antibodies labeled with fluorescent dyes and biotin are extensively used in imaging, immunoassays, flow cytometry, western blots, and immunoprecipitation among other applications3-6. Radiolabeled antibodies3,7 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 steps9-12. 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 internalization13-16. 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 proposed17,18. 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 (&#62;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.

<table border="0" cellpadding="0" cellspacing="0" width="592">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein A Beads</td>
			<td style="width:103px;">Promega Corporation</td>
			<td style="width:119px;">&#160;G8781</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein G Beads</td>
			<td style="width:103px;">Promega Corporation&#160;</td>
			<td style="width:119px;">&#160;G7471</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-SE (Succinimidyl Ester)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-ME (Malemide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A10255</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 647-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20347</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescein-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">F-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic Stand</td>
			<td style="width:103px;">Promega</td>
			<td style="width:119px;">Z5332</td>
			<td></td>
		</tr>
	</tbody>
</table>

antibody labeling with fluorescent dyes using magnetic protein a and protein g beads

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.

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...

Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

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

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.

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

The overall goal of this method is to label antibodies with fluorescent dies from purified samples or directly from cell medium using magnetic protein A or G beads. This method can be used to optimize the labeling chemistry of even small amount of antibodies within the cell medium. Leading to better downstream application of the antibodies.The main advantages of this technique are that the antibodies do not require purification, amine or thiol chemistry can be used, and the method can be automated or manual. Begin by uniformly re-suspending the beads with gentle shaking. Then, add 50 microliters of beads to a 1.5 milliliter microsentrifuge tube.Then, place the tube in a magnetic stand. After 10 seconds, carefully replace the storage buffer with 250 microliters of antibody-binding buffer and mix the beads well. After another 10 seconds, carefully replace the binding buffer with one milliliter of the antibody of interest.And mix the sample for 60 minutes at room temperature. At the end of the incubation, return the tubes to the magnet and remove the supernatant when the beads have moved to the sides of the tube. Then, rinse the beads and antibody mixture by two 10 second washes with 250 microliters of antibody-binding wash buffer per wash.To label the antibodies using amine chemistry, after the second wash, replace the wash buffer with 100 microliters of amine conjugation buffer and add 2.5 microliters of freshly prepared amine-reactive dye per 100 micrograms of anitbody. Vortex the sample for 60 minutes at room temperature to keep the beads in suspension during the labeling. Then, return the tube to the magnet for 10 seconds and remove the supernatant.Wash the beads two times with antibody-binding wash buffer, as just demonstrated. Then, replace the wash buffer with 50 microliters of elution buffer and mix the beads for five minutes. Return the tube to the magnet for 10 seconds and transfer the eluted antibody to a new microcentrifuge tube containing five microliters of neutralization buffer.Elute the antibody one more time, as just demonstrated, pulling the eluted samples. Then, measure the absorbance of the antibody-dye conjugate at 280 nanometers and at the lambda max for the dye. To label the antibodies using thiol chemistry, after the second wash, replace the wash buffer with 250 microliters of thiol conjugation buffer and mix the beads with gentle pipetting.After 10 seconds, wash the beads two times with antibody-binding wash buffer. Then, replace the wash buffer with 100 microliters of thiol conjugation buffer and add DTT to a final concentration of 2.5 millimolar. Mix the beads for another 60 minutes.Then, return the beads to the magnet for 10 seconds and discard the buffer. Next, add 250 microliters of thiol-conjugation buffer to the beads with gentle mixing. After 10 seconds, wash the beads two times with antibody-binding wash buffer, replacing the second wash with 100 microliters of fresh thiol-conjugation buffer.Now, add 2.5 microliters of freshly prepared thiol-reactive dye per 100 micrograms of antibody and mix the beads for 60 minutes at room temperature. At the end of the incubation, return the tube to the magnet for 10 seconds. And replace the supernatant with 250 microliters of fresh thiol-conjugation buffer with gentle mixing.After 10 seconds, wash the beads two times. Then, replace the wash buffer with 50 microliters of elution buffer and elute the conjugated antibody two times, as just demonstrated. Then, measure the absorbance of the antibody-dye conjugate at 280 nanometers and at the lambda max for the dye.The strong affinity between the antibodies and Protein G, the robust affinity of the magnet for the beads, and the optimization of the method prior to the bead purification together result in a nominal loss of antibody during the labeling reaction, as evidenced by the efficient recovery of three different mouse antibody isotypes after fluorescent dilabeling compared to simple purification. This recovery is also evident in Coomassie gels, where the low non-specific binding properties of the magnetic G Protein beads result in the presence of highly purified fluorescence-dilabeled heavy and light chains. On bead labeling with magnetic Protein A beads, it is also compatible with multiple fluorescent dyes, resulting in high recovery and good dye-to-antibody ratios.Once mastered, this technique can be completed in two to three hours, if it is performed properly. Allowing multiple samples to be handled manually or an automated platform for improved throughput. While attempting this procedure, it is important to remember to keep the magnetic beads in suspension at all times.Choose the appropriate volume of magnetic beads for the sample. And optimize the amount of fluorescent dye for the antibody of interest. Following this procedure, the labeled antibodies should be tested to determine functionality using relevant biological assays, like ELISA, facts, or cell-based assays.After watching this video, you should have a good understanding of how to label purified and unpurified antibodies with fluorescent tags or other small molecules, like biotin or cytotoxic drugs, using magnetic Protein A or G beads.

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JoVE Journal

Immunology and Infection

11.6K visualizações.  Promega Corporation. The overall goal of this method is to label antibodies with fluorescent dies from purified samples or directly from cell medium using magnetic protein A or G beads. This method can be used to optimize the labeling chemistry of even small amount of antibodies within the cell medium. Leading to better downstream application of the antibodies.The main advantages of this technique are that the antibodies do not require purification, amine or thiol chemistry can be used, and the method can ...