Affinity Chromatography pp 7-23

Part of the Methods in Molecular Biology book series (MIMB, volume 147)

Weak Affinity Chromatography

  • Magnus Strandh
  • HÅkan S. Andersson
  • Sten Ohlson


Since the inception of affinity chromatography 30 years ago (1), it has developed into a powerful tool mainly for the purification of proteins. It is based on the reversible formation of a tight binding complex between a ligand, immobilized on an insoluble matrix and a substance, the ligate, to be isolated from the solution. Typically the ligate is adsorbed by a column with the immobilized ligand, whereas noninteracting substances are washed off. By changing the elution conditions, the ligate can be released in a highly purified form. Some researchers argue that this procedure is based on specific extraction rather than by chromatography, which should rely on the differential migration of various substances. Regardless of the definitions, it is clear that traditional affinity chromatography exploits high affinity or avidity (binding constant (Ka) > 105/M) between the interacting molecules, which will result in an effective adsorption of the ligate. In this context the distinction between affinity and avidity is important: Whereas affinity describes the interaction in an individual binding site, avidity describes the multivalent binding between multiple binding sites of the ligand and ligate, respectively. High binding strength is required to achieve efficient adsorption, whereas weaker interactions will not produce adequate binding and therefore insufficient specificity will be acquired. This statement that strong specific binding is a prerequisite for the successful isolation of an interacting molecule has been in a nutshell the consensus of affinity chromatography.


  1. 1.
    Cuatrecasas P., Wilchek M., and Anfinsen C. B. (1968) Selective enzyme purification by affinity chromatography. Proc. Natl. Acad. Sci. USA 61, 636–643.PubMedCrossRefGoogle Scholar
  2. 2.
    Wikstrom M. and Ohlson S. (1992) Computer simulation of weak affinity chromatography. J. Chromatogr. 597, 83–92.CrossRefGoogle Scholar
  3. 3.
    Kucera E. (1965) Contribution to the theory of chromatography linear non-equilibrium elution chromatography. J. Chromatogr. 19, 237–248.PubMedCrossRefGoogle Scholar
  4. 4.
    Wade J. L., Bergold A. F., and Carr P. W. (1987) Theoretical description of nonlinear chromatography, with applications to physicochemical measurements in affinity chromatography and implications for preparative-scale separations. Anal. Chem. 59, 1286–1295.CrossRefGoogle Scholar
  5. 5.
    Fairchild P. J. and Wraith D. C. (1996) Lowering the tone: mechanisms of immunodominance among epitopes with low affinity for MHC. Immunol. Today 17, 80–85.PubMedCrossRefGoogle Scholar
  6. 6.
    Haywood A. M. (1994) Virus receptors: Binding, adhesion strengthening, and changes in viral structure. J. Virol. 68, 1–5.PubMedGoogle Scholar
  7. 7.
    Hakomori S.-I. (1993) Structure and function of sphingoglycolipids in transmem-brane signaling and cell-cell interactions. Biochem. Soc. Trans. 21, 583–595.PubMedGoogle Scholar
  8. 8.
    van der Merwe P. A., Brown M. H., Davis. S. J., and Barclay A. N. (1993) Affinity and kinetic analysis of the interaction of the cell adhesion molecules rat CD2 and CD48. EMBO J. 12, 4945–4954.PubMedGoogle Scholar
  9. 9.
    Reilly P. L., Woska Jr. J. R., Jeanfavre D. D., McNally E., Rothlein R., and Bormann B.-J. (1995) The native structure of intercellular adhesion molecule-1 (ICAM-1) is a dimer. J. Immunol. 155, 529–532.PubMedGoogle Scholar
  10. 10.
    Ohlson S., Lundblad A., and Zopf D. (1988) Novel approach to affinity chroma-tography using “weak” monoclonal antibodies. Anal. Biochem. 169, 204–208.PubMedCrossRefGoogle Scholar
  11. 11.
    Zopf D. and Ohlson S. (1990) Weak-affinity chromatography. Nature 346, 87–88.CrossRefGoogle Scholar
  12. 12.
    Schittny J. C. (1994) Affinity retardation chromatography: characterization of the method and its application. Anal. Biochem. 222, 140–148.PubMedCrossRefGoogle Scholar
  13. 13.
    Ohlson S., Bergstrom M., Pahlsson P., and Lundblad A. (1997) Use of monoclonal antibodies for weak affinity chromatography. J. Chromatogr. A 758, 199–208.PubMedCrossRefGoogle Scholar
  14. 14.
    Strandh M., Ohlin M., Borrebaeck C. A. K., and Ohlson S. (1998) New approach to steroid separation based on a low affinity IgM antibody. J. Immunol. Methods 214, 73–79.PubMedCrossRefGoogle Scholar
  15. 15.
    Chaiken I. M., Rosé S., and Karlsson R. (1992) Analysis of macromolecular interactions using immobilized ligands. Anal. Biochem. 201, 197–210.PubMedCrossRefGoogle Scholar
  16. 16.
    Fassina G., Zamai M., Brigham-Burke M., and Chaiken I. M. (1989) Recognition properties of antisense peptides to Arg8-vasopressin/bovine neurophysin 2 biosynthetic precursor sequences. Biochemistry 28, 8811–8818.PubMedCrossRefGoogle Scholar
  17. 17.
    Kauvar L. M., Cheung P. Y. K., Gomer R. H., and Fleischer A. A. (1990) Paralog chromatography. BioChromatography 5, 22–26.Google Scholar
  18. 18.
    Lu F. X., Aiyar N., and Chaiken I. M. (1991) Affinity capture of Arg8-vasopressin-receptor using immobilized antisense peptide. Proc. Natl. Acad. Sci. USA 88, 3637–3641.CrossRefGoogle Scholar
  19. 19.
    Pingali A., McGuinness B., Keshishian H., Fei-Wu J., Varady L., and Regnier F. E. (1996) Peptides as affinity surfaces for protein purification. J. Mol. Recogn. 9, 426–432.CrossRefGoogle Scholar
  20. 20.
    Ohlson S. and Zopf D. (1993) Weak affinity chromatography, in Handbook of Affinity Chromatography vol. 63: Chromatographic Science Series, (Kline, T., ed.), Marcel Dekker, Inc., New York, pp. 299–314.Google Scholar
  21. 21.
    Tsuji T., Yamamoto K., and Osawa T. (1993) Affinity chromatography of oli-gosaccharides and glycopeptides with immobilized lectins, in Molecular Interactions in Bioseparations (Ngo, T. T., ed.), Plenum, New York, pp. 113–126.Google Scholar
  22. 22.
    Leickt L., Bergström M., Zopf D., and Ohlson S. (1997) Bioaffinity chromatography in the 10 mM range of Kd. Anal. Biochem. 253, 135,136.PubMedCrossRefGoogle Scholar
  23. 23.
    Yang Q. and Lundahl P. (1995) Immobilized proteoliposome affinity chro-matography for quantitative analysis of specific interactions between solutes and membrane proteins. Interaction of cytochalasin B and D-glucose with the glucose transporter Glutl. Biochemistry 34, 7289–7294.PubMedCrossRefGoogle Scholar
  24. 24.
    Armstrong D., Ward T., Armstrong R., and Beesley T. (1986) Separation of drug stereoisomers by the formation of p-cyclodextrin inclusion complexes. Science 232, 1132–1135.PubMedCrossRefGoogle Scholar
  25. 25.
    Allenmark S and Andersson S. (1993) Chromatographic resolution of chiral compounds by means of immobilized proteins, in Molecular Interactions in Bioseparations (Ngo T. T., ed.), Plenum, New York, pp. 179–187.Google Scholar
  26. 26.
    Loun B. and Hage D. S. (1995) Chiral separation mechanisms in immobilized protein affinity columns: Binding of R-and S-warfarin to human serum albumin. J. Mol. Recogn. 8, 235.Google Scholar
  27. 27.
    Perrin S. R. and Pirkle W. H. (1991) Commercially available brush-type chiral selectors for the direct resolution of enantiomers. ACS Symp. Ser. 471, 43–66.CrossRefGoogle Scholar
  28. 28.
    Kempe M. and Mosbach K. (1991) Binding studies on substrate-and enantio-selective molecularly imprinted polymers. Anal. Lett. 24, 1137–1145.Google Scholar
  29. 29.
    Andersson H. S., Koch-Schmidt A.-C., Ohlson S., and Mosbach K. (1996) Study of the nature of recognition in molecularly imprinted polymers. J. Mol. Recogn. 9, 675–682.CrossRefGoogle Scholar
  30. 30.
    Ohlson S., Hansson L., Larsson P.-O., and Mosbach K. (1978) High performance liquid affinity chromatography (HPLAC) and its application to the separation of enzymes and antigens. FEBS Lett. 93, 5–9.PubMedCrossRefGoogle Scholar
  31. 31.
    Clonis Y. D. (1992) High performance liquid affinity chromatography for protein separation and purification, in Practical Protein Chromatography. Methods in Molecular Biology (Kenney A. and Fowell S., eds.), Humana Press, Totowa, NJ, pp. 105–124.Google Scholar
  32. 32.
    Griffiths A., Williams S., Hartley O., Tomlinson I., Waterhouse P., Crosby W., Kontermann R., Jones P., Low N., Allison T., Prospero T., Hoogenboom H., Nissim A., Cox J., Harrison J., Zaccolo M., Gherardi E., and Winter G. (1994) Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13, 3245–3260.PubMedGoogle Scholar
  33. 33.
    Hermanson G. T., Mallia A. K., and Smith P. K., eds. (1992) Immobilized Affinity Ligand Techniques. Academic Press, San Diego, CA.Google Scholar
  34. 34.
    Hayden M. S., Gilliland L. K., and Ledbetter J. A. (1997) Antibody engineering. Curr. Opin. Immunol. 9, 201–212.PubMedCrossRefGoogle Scholar
  35. 35.
    Smith G. and Petrenko V. (1997) Phage display. Chem. Rev. 97, 391–410.Google Scholar
  36. 36.
    Zopf D., Levinson R. E., and Lundblad A. (1982) Determination of a glucose-containing tetrasaccharide in urine by radioimmunoassay. J. Immunol. Methods 48, 109–119.PubMedCrossRefGoogle Scholar
  37. 37.
    Mudgett-Hunter M., Margolies M. N., Ju A., and Haber E. (1982) High-affinity monoclonal antibodies to the cardiac glycoside, digoxin. J. Immunol. 129, 1165–1172.Google Scholar
  38. 38.
    Lundblad A., Schroer K., and Zopf D. (1984) Radioimmunoassay of a glucose-containing tetrasaccharide using a monoclonal antibody. J. Immunol. Methods 68, 217–226.PubMedCrossRefGoogle Scholar
  39. 39.
    Danielsson L., Furebring C., Ohlin M., Hultman L., Abrahamson M., Carlsson R., and Borrebaeck C. (1991) Human monoclonal antibodies with different fine specificity for digoxin derivatives: cloning of heavy and light chain variable region sequences. Immunology 74, 50–54.PubMedGoogle Scholar
  40. 40.
    Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  41. 41.
    Ljungberg H., Ohlson S., and Nilsson S. (1998) Exploitation of a monoclonal antibody for weak affinity based separation in capillary gel electrophoresis. Elec-trophoresis 19, 461–464.CrossRefGoogle Scholar
  42. 42.
    Jonsson U. (1991) Real-time biospecific interaction analysis using surface plas-mon resonance and a sensor chip technology. BioTechniques 11, 620–627.PubMedGoogle Scholar
  43. 43.
    Ohlson S., Strandh M., and Nilshans H. (1997) Detection and characterization of weak affinity antibody-antigen recognition with biomolecular interaction analysis. J. Mol. Recogn. 10, 135–138.CrossRefGoogle Scholar
  44. 44.
    Strandh M., Persson B., Roos H., and Ohlson S. (1998) Studies of interactions with weak affinities and low molecule weight compounds using surface plasmon resonance technology. J. Mol. Recogn. 11, 188–190.CrossRefGoogle Scholar
  45. 45.
    Leickt L., Grubb A., and Ohlson S. (1998) Screening for weak monoclonal antibodies in hybridoma technology. J. Mol. Recogn. 11, 114–116.CrossRefGoogle Scholar
  46. 46.
    Akerstrom B., Brodin T., Reis K., and Bjorck L. (1985) Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol. 135, 2589–2592.PubMedGoogle Scholar
  47. 47.
    Kellogg D. R. and Alberts B. M. (1992) Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies. Mol. Biol. Cell 3, 1–11.PubMedGoogle Scholar
  48. 48.
    O’Shannessy D. J. and Wilchek M. (1990) Immobilization of glycoconjugates by their oligosaccharides: use of hydrazido-derivatized matrixes. Anal. Biochem. 191, 1–8.PubMedCrossRefGoogle Scholar
  49. 49.
    Ohlson S. (1992) Exploiting weak affinities, in Practical Protein Chromatography. Methods in Molecular Biology. (Kenney A. and Fowell S., eds.), Humana, Totowa, NJ, pp. 197–208.Google Scholar
  50. 50.
    Kasai K.-I., Oda Y., Nishikata M., and Ishii S.-I. (1986) Frontal affinity chromatography: theory for its application to studies on specific interactions of biomolecules. J. Chromatogr. 376, 33–47.PubMedCrossRefGoogle Scholar
  51. 51.
    Majors R. (1972) High performance liquid chromatography on small particle silica gel. Anal. Chem. 44, 1722–1726.CrossRefGoogle Scholar
  52. 52.
    Lawing A., Lindstrom L., and Grill C. (1992) An improved procedure for packing annular expansion preparative HPLC columns. The use of surfactants in the packing slurry. LC GC-Mag. Separation Sci. 10, 778–781.Google Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Magnus Strandh
  • HÅkan S. Andersson
  • Sten Ohlson
    • 1
  1. 1.Department of Natural SciencesUniversity of KalmarKalmarSweden

Personalised recommendations