Antibody Conjugates for Cell Biology

Rosaria P. Haugland1

1 Molecular Probes, Inc., Eugene, Oregon
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 16.5
DOI:  10.1002/0471143030.cb1605s06
Online Posting Date:  May, 2001
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Abstract

Antibody conjugates are extremely useful reagents for probing many biologically and chemically important molecules in vitro or in vivo. This unit presents some basic protocols for conjugating antibodies with fluorescent dyes, with biotin, and with enzymes. These protocols have been selected because they are relatively easy to perform and have high rates of success. The basic approach for conjugating antibodies is to derivatize amino groups on the antibody with the dye or biotin derivative of choice. Conjugation at amine sites is simple to perform, and, with a fluorescent dye, it generally yields brightly fluorescent conjugates. The conjugates retain high levels of biological activity, notwithstanding the fact that the labels react with random amines on the antibody molecule, and they may even react near or at the antigenā€binding site. Labeling of the amines is the most widely used commercial method to tag antibodies. In this unit, suggestions are presented for variations on these basic protocols that are useful when quantities of available antibody are limited. Methods for estimating antibody concentration are also included.

     
 
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Table of Contents

  • Basic Protocol 1: Conjugating Antibodies to Fluorophores or Biotin
  • Alternate Protocol 1: Conjugation of Flurophores or Biotin with F(ab′)2 or Fab Antibody Fragments
  • Support Protocol 1: Methods to Estimate Antibody Concentration
  • Basic Protocol 2: Conjugation of Antibodies with Enzymes
  • Alternate Protocol 2: Conjugation at the Carbohydrate Site of the Antibody
  • Alternate Protocol 3: Conjugation at the Carbohydrate Site of the Enzyme
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Conjugating Antibodies to Fluorophores or Biotin

  Materials
  • Antibody to label
  • 1.0 M sodium bicarbonate (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Probe (fluorophore or biotin)
  • Anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO; see recipe)
  • 1.5 M hydroxylamine, pH 8.0 (see recipe)
  • Size‐exclusion chromatography matrix: MATREX Cellufine GH25 (Amicon/Millipore), BioGel P30 (BioRad Laboratories) or equivalent matrix
  • ToyoPearl HW‐40F (TosoHaas)
  • Reaction buffer, pH 7.5 (see recipe)
  • Appropriate TLC solvent (see Table 16.5.1, optional)
    Table 6.5.1   Materials   TLC Solvents and Suggested Incubation Molar Ratios of Probe to Antibody for Several Commonly Used Fluorophores a   TLC Solvents and Suggested Incubation Molar Ratios of Probe to Antibody for Several Commonly Used Fluorophores

    Fluorophore MR (Ab at 1‐3 mg/ml) MR (Ab at 4‐10 mg/ml) MW (Da) Probe (mg/ml) TLC solvent
    Alexa Fluor 350 15‐25 10‐15 410 10 H 2O:AcCN :: 20:80
    Alexa Fluor 430 15‐20 8‐10 623 10 H 2O:AcCN :: 20:80
    Alexa Fluor 488 20‐25 15‐20 643 10 H 2O:AcCN :: 20:80
    Alexa Fluor 532 14‐18 10‐12 724 10 H 2O:AcCN :: 20:80
    Alexa Fluor 546 12‐15 8‐10 1079 10 H 2O:AcCN :: 20:80
    Alexa Fluor 568 25‐30 10‐20 792 10 H 2O:AcCN :: 20:80
    Alexa Fluor 594 20‐30 10‐20 819 10 H 2O:AcCN :: 20:80
    BODIPY FL 15‐30 10‐15 641 10 C:M:A :: 70:25:5
    Cascade Blue 15‐20 10‐15 607 10 D:H 2O:P:NH 4 :: 50:16:19:15
    Cy3 Bis SE 15‐28 5‐15 944 10 C:M:A :: 70:25:5
    Cy5 Mono SE 10‐20 5‐10 792 10 C:M:A :: 70:25:5
    Eosin 50‐70 40‐60 705 10 C:M:A :: 70:25:5
    FITC 40‐60 30‐60 389 10 C:M:A :: 70:25:5
    Fluorescein‐EX 20‐25 15‐20 590 10 C:M:A :: 70:25:5
    Marina Blue 8‐12 6‐10 367 10 C:M:A :: 70:25:5
    Oregon Green 488 15‐20 10‐15 509 10 C:M:A :: 70:25:5
    Oregon Green 514 15‐20 10‐15 609 10 C:M:A :: 70:25:5
    Rhodamine Red‐X 8‐10 5‐8 771 5 C:M :: 85:15
    Rhodol Green 10‐15 5‐10 472 10 C:M:A :: 70:25:5
    Tetramethyl‐rhodamine (TMR) 15‐20 8‐10 528 10 C:M:A :: 70:25:5
    Texas Red‐X 5‐8 5‐8 817 5 C:M:A :: 70:25:5
    Biotin
    Biotin‐XX 20‐25 10‐20 568 10

     aMR = Incubation molar ratio of fluorophore to antibody, when antibody is at 1 to 3 mg/ml (column 2) or at 4‐10 mg/ml (column 3); SE = succinimidyl ester. Solvents: C = chloroform; M = methanol; A = acetic acid; D = dioxane; P = isopropyl alcohol; NH 4 = ammonia; AcCN = acetonitrile.
  • 10 mM 4′‐hydroxyazobenzene‐2‐carboxylic acid (HABA) in 10 mM NaOH (for biotinylation)
  • Assay buffer: 50 mM sodium phosphate/150 mM NaCl, pH 6.0 (for biotinylation)
  • 0.5 mg/ml avidin in assay buffer (for biotinylation)
  • 0.25 mM biotin in assay buffer (for biotinylation)
  • Thin layer chromatography (TLC) silica gel/aluminum plates (EM Science; optional)
  • TLC chamber (optional)
  • Dialysis tubing
  • Chromatography column of appropriate size (see step )
  • Ultrafrel centrifugal filter devices, Biomax‐50K or‐100K (Amicon/Millipore; optional)
  • Additional reagents and equipment for size exclusion chromatography (unit 5.5) and dialysis ( appendix 3C)

Alternate Protocol 1: Conjugation of Flurophores or Biotin with F(ab′)2 or Fab Antibody Fragments

  Materials
  • HRPO, APase, GO, βGase (Boehringer Mannheim, Sigma)
  • Reaction buffer, pH 7.5 (see recipe)
  • TEA buffer (see recipe; for APase only)
  • Succinimidyl 3‐(2‐pyridyldithio)propionate (SPDP, MW 312)
  • Anhydrous dimethylsulfoxide (DMSO) or dimethylformamide (DMF)
  • BIO‐GEL P‐30 (Bio‐Rad) or Cellufine GH‐25 (Amicon/Millipore)
  • Dithiothreitol (DTT)
  • Antibody to label
  • Succinimidyl trans‐4‐(N‐maleimidylmethyl)cyclohexane‐1‐carboxylate (SMCC, MW 334)
  • Tris‐(2‐carboxyethyl)phosphine, hydrochloride (TCEP, MW 287; Molecular Probes, Pierce Chemical)
  • 50 mM N‐ethylmaleimide (MW 125.13)
  • Sephacryl S‐200 for HRPO conjugates (Amersham Pharmacia Biotech or Sigma Chemical)
  • BIO‐GEL A‐0.5m for APase conjugates (Bio‐Rad Laboratories)
  • BIO‐GEL A‐1.5m for BGase conjugates (Bio‐Rad Laboratories)
  • Chromatography column of appropriate size (see step )
  • Ultrafrel centrifugal filter devices, Biomax‐50K or‐100K (Amicon/Millipore)
  • Additional reagents and equipment for dialysis ( appendix 3C) and size exclusion chromatography (unit 5.5)

Support Protocol 1: Methods to Estimate Antibody Concentration

  Materials
  • Antibody to label in solution (see protocol 1, step )
  • 0.1 M acetate buffer, pH 6.0
  • 20 mM sodium metaperiodate in acetate buffer, pH 6.0, ice cold
  • Reaction buffer, pH 7.5 (see recipe)
  • 100 mM aqueous sodium cyanoborohydride, freshly prepared
  • HRPO, APase, βGase, GO
  • Column chromatography matrices (see protocol 4)
NOTE: Protect the sample from light during the entire procedure.
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Figures

Videos

Literature Cited

Literature Cited
   Alon, R., Bayer, E.A., and Wilchek, M. 1990. Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin. Biochem. Biophys. Res. Commun. 170:1236‐1241.
   Banks, P.R. and Paquette, D.M. 1995. Comparison of three common amine‐reactive fluorescent probes used for conjugation to biomolecules by capillary zone electrophoresis. Bioconj. Chem. 6:447‐458.
   Briggs, J. and Panfili, P.R. 1991. Quantitation of DNA and protein impurities in biopharmaceuticals. Anal. Chem. 63:850‐859.
   Carlsson, J., Drevin, H., and Axen, R. 1978. Protein thiolation and reversible protein‐protein conjugation. N‐Succinimidyl 3‐(2‐pyridyldithio) propionate, a new heterobifunctional reagent. Biochem. J. 173:723‐737.
   Duncan, R.J., Weston, P.D., and Wrigglesworth, R. 1983. A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132:68‐73.
   Getz, E.B., Xiao, M., Chakrabarty, T., Cooke, R., and Selvin, P.R. 1999. A comparison between the sulfhydryl reductants Tris(2‐carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal. Biochem. 273:73‐80.
   Green, N.M. 1975. Avidin. In Advances in Protein Chemistry (C.B. Anfinsen, J.T. Edsall, and F.M. Richards, eds.) pp. 85‐133. Academic Press, New York.
   Gretch, D.R., Suter, M., and Stinski, M.F. 1987. The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate Herpes virus hydrophobic proteins or glycoproteins. Anal. Chem. 163:270‐277.
   Hage, D.S., Wolfe, C.A., and Oates, M.R. 1997. Development of a kinetic model to describe the effective rate of antibody oxidation by periodate. Bioconj. Chem. 8:914‐920.
   Haugland, R.P. 1996a. Introduction to amine modification. In Handbook of Fluorescent Probes and Research Chemicals, 6th ed. (M.T.Z. Spence, ed.) p. 12. Molecular Probes, Eugene, Ore.
   Haugland, R.P. 1996b. Coupling of monoclonal antibodies with enzymes. In Methods in Molecular Biology (W.C. Davis, ed.) pp. 235‐243. Humana Press, Totowa, N.J.
   Haugland, R.P. 1996c. Biotins and haptens. In Handbook of Fluorescent Probes and Research Chemicals, 6th ed. (M.T.Z. Spence, ed.) pp. 82‐88. Molecular Probes, Eugene, Ore.
   Haugland, R.P. 1999. Handbook of Fluorescent Probes and Research Chemicals, 7th ed. (CD‐ROM). Molecular Probes, Eugene, Ore.
   Haugland, R.P. and You, W.W. 1998. Coupling of monoclonal antibodies with biotin. In Methods in Molecular Biology (W.C. Davis, ed.) pp. 173‐184. Humana Press, Totowa, N.J.
   Hermanson, G.T. 1996a. Preparation of antibody‐enzyme conjugates. In Bioconjugate Techniques, p. 486. Academic Press, San Diego.
   Hermanson, G.T. 1996b. Amine‐reactive chemical reactions. In Bioconjugate Techniques, pp. 139‐140. Academic Press, San Diego.
   Hermanson, G.T. 1996c. Preparation of antibody‐enzyme conjugates. In Bioconjugate Techniques, pp. 463‐465. Academic Press, San Diego.
   Hnatowich, D.J., Virzi, F., and Rusckowski, M. 1987. Investigations of avidin and biotin for imaging applications. J. Nucl. Med. 28:1294‐1302.
   Hoffman, K., Wood, S.W., Brinton, C.C., Montibeller, J.A., and Finn, F.M. 1980. Iminobiotin affinity columns and their application to retrieval of streptavidin. Proc. Natl. Acad. Sci. U.S.A. 77:4666‐4668.
   Husain, M. and Bieniarz, C. 1994. Fc site–specific labeling of immunoglobulins with calf intestinal alkaline phosphatase. Bioconj/ Chem. 5:482‐490.
   Johnson, I.D. 1996. Introduction to fluorescence techniques. In Handbook of Fluorescent Probes and Research Chemicals, 6th ed. (M.T.Z. Spence, ed.) pp. 1‐6. Molecular Probes, Eugene, Ore.
   Lefevre, C., Kang, H.C., Haugland, R.P., Malekzadeh, N., Arttamangkul, S., and Haugland, R.P. 1996. Texas Red‐X and Rhodamine Red‐X, new derivatives of sulforhodamine 101 and Lissamine rhodamine B with improved labeling and fluorescence properties. Bioconj.Chem. 7:482‐489.
   Morag, E., Bayer, E.A., and Wicheck, M. 1996. Reversibility of biotin‐binding by selective modification of tyrosine in avidin. Biochem. J. 316:193‐199.
   Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., and Waggoner, A.S. 1989. Cyanine dye labeling reagents containing isothiocyanate groups. Cytometry 10:1‐9.
   Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J., and Waggoner, A.S. 1993. Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconj. Chem. 4:105‐111.
   Mujumdar, S.R., Mujumdar, R.B., Grant, C.M., and Waggoner, A.S. 1996. Cyanine labeling reagents: Sulfobenzindocyanine succinimidyl esters. Bioconj. Chem. 7:356‐362.
   O'Malley, J.J. and Weaver, J.L. 1972. Subunit structure of glucose oxidase from Aspergillus niger. Biochemistry 12:3527‐3532.
   Orr, G.A. 1981. The use of the 2‐iminobiotin‐avidin interaction for the selective retrieval of labeled plasma membrane components. J. Biol. Chem. 256:761‐766.
   Panchuk‐Voloshina, N., Haugland, R.P., Bishop‐Stewart, J., Bhalgat, M.K., Millard, P.J., Mao, F., and Leung, W.Y. 1999. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47:1179‐1188.
   Ravdin, P. and Axelrod, D. 1977. Fluorescent tetramethylrhodamine derivatives of alpha‐bungarotoxin: Preparation, separation, and characterization. Anal. Biochem. 80:585‐592.
   Rodwell, J.D., Alvarez, V.L., Lee, C., Lopes, A.D., Goers, J.W., King, H.D., Powsner, H.J., and McKearn, T.J. 1986. Site‐specific covalent modification of monoclonal antibodies: In vitro and in vivo evaluations. Proc. Natl. Acad. Sci. U.S.A. 83:2632‐2636.
   Welinder, K.G. 1979. Amino acid sequence studies of horseradish peroxidase. Eur. J. Biochem. 96:483‐502.
   Whitaker, J.E., Haugland, R.P., Moore, P.L., Hewitt, P.C., Resse, M., and Haugland, R.P. 1991. Cascade Blue derivatives: Water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers. Anal. Biochem. 198:119‐130.
   Wilchek, M. and Bayer, E.A. 1990. Introduction to avidin‐biotin technology. Methods Enzymol. 184:5‐13.
   Wong, S.S. 1991. Reactive groups of proteins and modifying agents. In Chemistry of Protein Conjugation and Crosslinking, pp. 37. CRC Press, Boca Raton, Fla.
Key References
   Aslam, M. and Dent, A. 1998. Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences. Macmillan Reference, London.
  These two citations are excellent reference sources covering concepts behind all the procedures discussed in this unit.
   Hermanson, G.T. 1996. Bioconjugate Techniques. Academic Press, San Diego.
  Contains excellent background material for all procedures, as well as chemical structures, spectra, and other product information for many of the probes discussed in this article.
   Haugland, 1996b, 1999. See above. (The 7th edition of this Handbook (1999) is available free on CD‐ROM from Molecular Probes and at www.probes.com).
Internet Resources
   http://www.probes.com
  This Molecular Probes site is a searchable, complete handbook and catalog of fluorescent probes, protein conjugates, and other probes for research. It contains downloadable spectra, structures, images, product information sheets, material safety data sheets, full scientific references, and link to contact the manufacturer.
   http://www.piercenet.com
  This Pieerce Chemical Company site is also a searchable handbook and catalog with technical tips and selection guides, literature, application notes, and link to contact the manufacturer.
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