Preparation of Avidin Conjugates

  • Rosaria P. Haugland
  • Mahesh K. Bhalgat
Part of the Springer Protocols Handbooks book series (SPH)


The high-affinity avidin-biotin system has found applications in different fields of biotechnology including immunoassays, histochemistry, affinity chromatography, and drug delivery, to name a few. A brief description of avidin and avidin-like molecules, streptavidin, deglycosylated avidin, and NeutrAvidin™ avidin, is presented in  Chapter 48. With four biotin binding sites per molecule, the avidin family of proteins is capable of forming tight complexes with one or more biotinylated compounds (1). Typically, the avidin-biotin system is used to prepare signal-amplifying ”sandwich“ complexes between specificity reagents (e.g., antibodies) and detection reagents (e.g., fluorophores, enzymes, and so on). The specificity and detection reagents are independently conjugated, one with avidin and the other with biotin, or both with biotin, providing synthetic flexibility (2).


Succinic Anhydride Biotin Binding Thioether Bond Affinity Matrice Biotin Binding Site 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Green, N. M. (1975) Avidin, in Advances in Protein Chemistry, Vol. 29 Anfinsen, C. M., Edsall, J. T., and Richards, F. M., eds.), Academic Press, New York, pp. 85–133.Google Scholar
  2. 2.
    Bayer, E. A. and Wilchek, M. (1980) The use of the avidin-biotin complex as a tool in molecular biology. Meth. Biochem. Analyt. 26, 1–45.CrossRefGoogle Scholar
  3. 3.
    Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Oligonucleotide-) directed self-assembly of proteins: semisynthetic DNA-streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates. Nucleic Acids Res. 22, 5330–5339.CrossRefGoogle Scholar
  4. 4.
    Paganelli, G., Belloni, C., Magnani, P., Zito, F., Pasini, A., Sassi, I., et al. (1992) Two-step tumor targetting in ovarian cancer patients using biotinylated monoclonal antibodies and radioactive streptavidin. Eur. J. Nucl. Med. 19, 322–329.PubMedCrossRefGoogle Scholar
  5. 5.
    van Osdol, W. W., Sung, C., Dedrick, R. L., and Weinstein, J. N. (1993) A distributed pharmacokinetic model of two-step imaging and treatment protocols: application to streptavidin-conjugated monoclonal antibodies and radiolabeled biotin. J. Nucl. Med. 34, 1552–1564.PubMedGoogle Scholar
  6. 6.
    Markwell, M. A. K. (1982) A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labeling of antiserum. Analyt. Biochem. 125, 427–432.PubMedCrossRefGoogle Scholar
  7. 7.
    Salacinski, P. R. P., McLean, C., Sykes, J. E. C., Clement-Jones, V. V., and Lowry, P. J. (1981) Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6,-tetrachloro-3α,6α-diphenyl glycoluril (iodogen). Analyt. Biochem. 117, 136–146.PubMedCrossRefGoogle Scholar
  8. 8.
    Mock, D. M. (1990) Sequential solid-phase assay for biotin based on 125I-labeled avidin in Meth. Enzymol. 184, 224–233.PubMedCrossRefGoogle Scholar
  9. 9.
    Bolton, A. E. and Hunter, W. M. (1973) The labeling of proteins to high specific radioactivity by conjugation to an 125I-containing acylating agent. Applications to the radioimmu-noassay. Biochem. J. 133, 529–539.PubMedGoogle Scholar
  10. 10.
    Vaidyanathan, G., Affleck, D. J., and Zalutsky, M. R. (1993) Radioiodination of proteins using N-succinimidyl 4-hydroxy-3-iodobenzoate. Bioconjugate Chem. 4, 78–84.CrossRefGoogle Scholar
  11. 11.
    Vaidyanathan, G. and Zalutsky, M. R. (1990) Radioiodination of antibodies via N-succinimidyl 2,4-dimethoxy-3-(trialkylstannyl)benzoates. Bioconjugate Chem. 1, 387–393.CrossRefGoogle Scholar
  12. 12.
    Hylarides, M. D., Wilbur, D. S., Reed, M. W., Hadley, S. W., Schroeder, J. R. and Grant, L. M. (1991) Preparation and in vivo evaluation of an N-(p-[125I]iodophenethyl)maleimide-antibody conjugate. Bioconjugate Chem. 2, 435–440.CrossRefGoogle Scholar
  13. 13.
    Arano, Y., Wakisaka, K., Ohmomo, Y., Uezono, T., Mukai, T., Motonari, H., Shiono, et al. (1994) Maleimidoethyl 3-(tri-n-butylstannyl)hippurate: A useful radioiodination reagent for protein radiopharmaceuticals to enhance target selective radioactivity localization. J. Med. Chem. 37, 2609–2618.PubMedCrossRefGoogle Scholar
  14. 14.
    Willner, D., Trail, P. A., Hofstead, S. J., Dalton King, H., Lasch, S. J., Braslawsky, G. R., et al. (1993) (6-Maleimidocaproyl)hydrazone of doxorubicin-a new derivative for the preparation of immunoconjugates of doxorubicin. Bioconjugate Chem. 4, 521–527.CrossRefGoogle Scholar
  15. 15.
    Arnold, Jr., L. J. (1985) Polylysine-drug conjugates. Meth. Enzymol. 112, 270–285.PubMedCrossRefGoogle Scholar
  16. 16.
    Pietersz, G. A. and McKenzie, I. F. (1992) Antibody conjugates for the treatment of cancer. Immunol. Rev. 129, 57–80.PubMedCrossRefGoogle Scholar
  17. 17.
    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.PubMedGoogle Scholar
  18. 18.
    Dimroth, P. (1986) Preparation, characterization, and reconstitution of oxaloacetate decarboxylase from Klebsiella aerogenes, a sodium pump. Meth. Enzymol. 125, 530–540.PubMedCrossRefGoogle Scholar
  19. 19.
    Dean, P. D. G., Johnson, W. S., and Middle, F. S. (1985) Activation procedures. In Affinity chromatography. A practical approach, IRL Press, Washington, DC, 34–35.Google Scholar
  20. 20.
    Kohanski, R. A. and Lane, D. (1990) Monovalent avidin affinity columns. Meth. Enzymol. 184, 194–220.PubMedCrossRefGoogle Scholar
  21. 21.
    Chilkoti, A., Schwartz, B. L., Smith, R. D., Long, C. J. and Stayton, P. S. (1995) Engineered chimeric streptavidin tetramers as novel tools for bioseparations and drug delivery. Bio/Technology 13, 1198–1204.PubMedCrossRefGoogle Scholar
  22. 22.
    Morag, E., Bayer, E. A., and Wilchek, M. (1996) Immobilized Nitro-Avidin and Nitro-Streptavidin as Reusable Affinity Materials for Application in Avidin-Biotin Technology. Analyt. Biochem. 243, 257–263.PubMedCrossRefGoogle Scholar
  23. 23.
    Wong, S. S. (1991) Reactive groups of proteins and their modifying agents, in Chemistry of Protein Conjugation and Crosslinking, CRC, Boston, MA, 30.Google Scholar
  24. 24.
    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.PubMedGoogle Scholar
  25. 25.
    Haugland, R. P. (1995) Coupling of monoclonal antibodies with fluorophores. In Methods in Molecular Biology, (Ed. Davis, W. C.) Humana, Totowa, NJ, 45, 205–221.Google Scholar
  26. 26.
    Kronick, M. N. and Grossman, P. D. (1983) Immunoassay techniques with fluorescent phycobiliprotein conjugates. Clin. Chem. 29, 1582–1586.PubMedGoogle Scholar
  27. 27.
    Bongartz, J.-P., Aubertin, A.-M., Milhaud, P. G. and Lebleu, B. Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide. Nucleic Acids Res. 22, 4681–4688.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Rosaria P. Haugland
    • 1
  • Mahesh K. Bhalgat
    • 2
  1. 1.Molecular Probes Inc.Eugene
  2. 2.Molecular Probes, Inc.Eugene

Personalised recommendations