Conjugation of Fluorochromes to Monoclonal Antibodies

Kevin L. Holmes1, Larry M. Lantz1, Wesley Russ2

1 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 2 Kirkegaard & Perry Laboratories, Gaithersburg, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 4.2
DOI:  10.1002/0471142956.cy0402s00
Online Posting Date:  May, 2001
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Abstract

Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is probably the most widely used application of flow cytometry. This unit contains protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, it provides a procedure for preparing a PE‐Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications.

Keywords: flow cytometry; monoclonal antibodies; fluorochromes; antibody labeling.

     
 
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Table of Contents

  • Basic Protocol 1: Labeling Antibody with Fluorescein Isothiocyanate (FITC)
  • Basic Protocol 2: Labeling Antibody with Long‐Armed Biotin
  • Basic Protocol 3: Labeling with Texas Red–X
  • Basic Protocol 4: Labeling Antibody with Phycobiliproteins
  • Basic Protocol 5: Conjugation of Texas Red to R‐Phycoerythrin to Produce an Energy Transfer Fluorochrome
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Labeling Antibody with Fluorescein Isothiocyanate (FITC)

  Materials
  • 1 to 2 mg/ml purified monoclonal antibody
  • FITC labeling buffer (prepare ≤2 weeks before use; see recipe)
  • 5 mg/ml FITC, isomer I, in anhydrous dimethyl sulfoxide (DMSO)
  • Final dialysis buffer (see recipe)
  • Sephadex G‐25 column (Pharmacia Biotech PD‐10; optional)
  • Dialysis tubing

Basic Protocol 2: Labeling Antibody with Long‐Armed Biotin

  Materials
  • 1 to 2 mg/ml purified monoclonal antibody
  • Succinimide ester labeling buffer or IgM labeling buffer (see reciperecipes)
  • 10 mg/ml long‐armed biotin (Zymed) in anhydrous N,N‐dimethylformamide (DMF)
  • Dialysis tubing

Basic Protocol 3: Labeling with Texas Red–X

  Materials
  • 1 to 2 mg/ml purified monoclonal antibody
  • Succinimide ester labeling buffer (see recipe)
  • 5 mg/ml Texas Red–X succinimidyl ester (Molecular Probes) in N,N‐dimethylformamide (DMF)
  • Final dialysis buffer (see recipe)
  • Stabilizing buffer (see recipe)
  • Dialysis tubing
  • Sephadex G‐25 column (Pharmacia Biotech; optional)

Basic Protocol 4: Labeling Antibody with Phycobiliproteins

  Materials
  • 10 to 25 mg/ml phycoerythrin (PE; purchased as suspension in buffered ammonium sulfate solution)
  • Coupling buffer, pH 5.5 and 7.5 (see recipe)
  • Sulfhydryl addition reagent: N‐succinimidyl‐S‐acetylthioacetate (SATA; Calbiochem; store under nitrogen after opening)
  • Dimethylformamide (DMF)
  • Nitrogen
  • Deacetylation buffer (see recipe)
  • Heterobifunctional cross‐linker: γ‐maleimidobutyric acid N‐hydroxysuccinimide ester (GMBS; Calbiochem; store under nitrogen after opening)
  • Tetrahydrofuran (THF)
  • 0.1 mg/ml cysteine
  • Running buffer, degassed (see recipe)
  • Dialysis tubing
  • AcA 34 column (IBF Biotechnics)
  • Sephacryl S‐200 column (Pharmacia Biotech; optional)

Basic Protocol 5: Conjugation of Texas Red to R‐Phycoerythrin to Produce an Energy Transfer Fluorochrome

  Materials
  • 10 to 50 mg R‐phycoerythrin (R‐PE; purchased as suspension in buffered ammonium sulfate solution)
  • R‐PE dialysis buffer (prepared within 2 days of use; see recipe)
  • Conjugation buffers A and B (see recipe)
  • Texas Red–sulfonyl chloride (Molecular Probes)
  • N,N‐Dimethylformamide (DMF)
  • Glycine (ultrapure or ACS grade)
  • 0.5 M hydroxylamine⋅HCl, pH 7.2 (prepared as for deacetylation buffer, but without EDTA; see recipe)
  • Equilibration buffer (see recipe for HIC column buffers)
  • First‐wash buffer (see recipe for HIC column buffers)
  • Elution buffers A and B (see recipe for HIC column buffers)
  • Dialysis tubing
  • Sephadex G‐50 fine columns, one with ∼5 ml capacity and one larger (e.g., ∼50 ml capacity for labeling 10 mg of R‐PE)
  • Fraction collector
  • HIC (hydrophobic interaction column) TSK‐Gel Toyopearl Butyl 650M (e.g., 25 ml capacity for labeling 10 mg of R‐PE)
  • Gradient maker with 200‐ml capacity
  • UV monitor and chart recorder
  • Magnetic stir plate
  • 10‐ to 15‐ml glass test tube
  • Flea (small stir‐bar)
  • Peristaltic pump
  • Centrifuge and appropriate tubes (e.g., Beckman tabletop with 50‐ml conical tubes)
  • Spectrophotometer with quartz cuvettes
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Figures

Videos

Literature Cited

Literature Cited
   Andrew, S.M. and Titus, J.A. 1991. Dialysis and concentration of protein solutions. In Current Protocols in Immunology (J.E., Coligan, Kruisbeck, A.M., Margulies, D.H., Shevach, E.M., and Strober, W., eds.) pp. A.3H.1‐A.3H.2. John Wiley and Sons, New York.
   Blattler, W.A., Kuenzi, B.S., Lambert, J.M., and Senter, P.D. 1985. New heterobifunctional protein cross‐linking reagent that forms an acid‐ labile link. Biochemistry. 24:1517‐1524.
   Brinkley, M. 1992. A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross‐linking reagents. Bioconjugate Chem. 3:2‐13.
   Duncan, R.J.S., Weston, P.D., and Wrigglesworth, R. 1981. A new reagent which may be used to introduce sulfydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem.. 132:68‐73.
   Glazer, A.N. and Stryer, L. 1983. Fluorescent tandem phycobiliprotein conjugates. Biophys. J. 43:383‐386.
   Hashida, S., Imagawa, M., Inoue, S., Ruan, K‐H., and Ishikawa, E. 1984. More useful maleimide compounds for the conjugation of Fab′ to horseradish peroxidase through thiol groups in the hinge. J. Appl. Biochem. 6:56‐63.
   Kitagawa, T., Shimozono, T., Aikawa, T., Yoshida, T., and Nishimura, H. 1981. Preparation and characterization of hetero‐bifunctional cross‐ linking reagents for protein modification. Chem. Pharm. Bull. (Tokyo) 29:1130‐1135.
   Kronick, M.N. 1988. Phycobiliproteins as labels in immunoassay. In Nonisotopic Immunoassay. (T.T. Ngo, ed.) pp 163‐185. Plenum Press, NY.
   Tanimori, H., Ishikawa, F., and Kitagawa, T. 1983. A sandwich enzyme immunoassay of rabbit immunoglobulin G with an enzyme labeling method and a new solid support. J. Immunol. Methods 62:123‐131.
   Titus, J.A., Haugland, R., Sharrow, S.O., and Segal, D.M. 1982. Texas Red, a hydrophilic, red‐emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J. Immunol. Methods 50:193‐204.
Key Reference
   Brinkley, 1992. See above.
  A good reference book for both protein‐protein coupling and reactive group chemistry
   Wong, S.S. 1991. Chemistry of Protein Conjugation and Cross‐Linking. CRC Press, Inc.
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