Cytometry > Probes

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

Conjugation of Fluorochromes to Monoclonal Antibodies

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

1National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
2Kirkegaard & 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
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Unit Introduction
  • 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
  • Bibliography
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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 recipes)
  • 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: -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
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 4.2.1
    Elution profile of phycoerythrin-labeled IgG from a gel filtration column. The initial peaks have greater than a 1:1 ratio of dye bound to antibody, and are not used. Optimal material is found in the center fractions designated 1:1. The trailing peaks contain unbound dye and unconjugated antibodies, and are discarded.

    View Image

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.
    Wong, S.S. 1991. Chemistry of Protein Conjugation and Cross-Linking. CRC Press, Inc.

A good reference book for both protein-protein coupling and reactive group chemistry

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.