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.
Promega Corporation; nidhi.nath@promega.com.
Nidhi
N
Nath
Promega Corporation.
Becky
B
Godat
Promega Corporation.
Marjeta
M
Urh
D000818:Animals; D000906:Antibodies; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D018796:Immunoconjugates; D018189:Immunomagnetic Separation / Q000379:methods*; D051379:Mice; D013205:Staphylococcal Protein A
D000906:Antibodies; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; C034284:IgG Fc-binding protein, Streptococcus; D018796:Immunoconjugates; D013205:Staphylococcal Protein A
Semin Oncol. 2014 Oct;41(5):613-22
25440606
Curr Opin Chem Biol. 2010 Dec;14(6):803-9
20952245
Methods Mol Biol. 2010;588:319-26
20012842
PLoS One. 2015 Apr 20;10(4):e0124708
25894652
J Immunol Methods. 2007 Apr 30;322(1-2):40-9
17383674
J Mol Recognit. 2011 Nov-Dec;24(6):945-52
22038801
Chem Biol Drug Des. 2013 Jan;81(1):113-21
23253133
Anal Bioanal Chem. 2012 Sep;404(5):1449-63
22797718
J Biomol Screen. 2016 Jan;21(1):12-23
26518032
Hematology Am Soc Hematol Educ Program. 2013;2013:306-10
24319196
J Mol Recognit. 2004 May-Jun;17(3):268-76
15137036
Biomagn Res Technol. 2004 Nov 26;2(1):7
15566570
Appl Microbiol Biotechnol. 2013 Nov;97(21):9343-53
24057405
Proteomics. 2014 Jan;14(1):14-8
24307663
J Immunol Methods. 2015 Nov;426:95-103
26316179
Nat Immunol. 2004 Dec;5(12):1211-7
15549122
Nat Methods. 2011 May 15;8(7):551-8
21572409
J Immunol Methods. 2016 Apr;431:11-21
26851520
MAbs. 2012 May-Jun;4(3):362-72
22531451
J Vis Exp. 2014 Nov 20;(93):e52099
25490604
Ann N Y Acad Sci. 2014 Aug;1321:41-54
25123209
Anal Biochem. 2010 Jul 15;402(2):146-50
20362543
Nat Biotechnol. 2012 Jan 22;30(2):184-9
22267010
ACS Chem Biol. 2014 Oct 17;9(10):2237-41
25140805