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Surface-Density and Orientation Effects on Immobilized Antibodies and Antibody Fragments

Bio/Technology volume 11, pages 825–829 (1993)Cite this article

Abstract

The binding parameters of randomly immobilized protein 315 and Fv fragments, as well as site-specifically immobilized Fab′ fragments, have been measured for a small hapten (MW=341 Daltons) and a large synthetic antigen (MW=50 kD). Immobilized Fv fragments had the highest binding capacities; hence, removing unnecessary protein domains can be beneficial for improving the total capacity of an immunosorbent. For all immunosorbents, high protein loadings led to relatively low specific activities (n values). This effect was reversible, however, as the loss of immobilized antibody upon prolonged storage partially restored the specific activity. At high loadings the specific activity of immobilized whole antibody was lower for the large antigen than for the small hapten, whereas no effect of hapten size on n was evident for either immobilized Fab′ or Fv fragments. Although a fraction of immobilized antibody was inactive at the higher loadings, EPR spectroscopy revealed no significant changes in the conformation of active immobilized antibody.

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References

  1. Janson, J.-C. 1984. Large-scale affinity purification—state of the art and future prospects. Trends Biotechnol. 2: 31–38.

    Article  CAS  Google Scholar 

  2. Chase, H.A. 1984. Affinity separations utilising immobilised monoclonal antibodies—a new tool for the biochemical engineer. Chem. Eng. Sci. 39: 1099–1125.

    Article  CAS  Google Scholar 

  3. Klausner, A. 1987. Immunoassays flourish in new markets. Bio/Technology 5: 551–56.

    Google Scholar 

  4. Carlsson, R., Glad, C. and Borrebaeck, C.K.A. 1989. Monoclonal antibodies into the '90s: the all-purpose tool. Bio/Technology 7: 567–73.

    CAS  Google Scholar 

  5. Scott, M.G. 1985. Monoclonal antibodies—approaching adolescence in diagnostic immunoassays. Trends Biotechnol. 3: 170–75.

    Article  Google Scholar 

  6. Lord, J.M., Roberts, L.M., Thorpe, P.E. and Vitetta, E.S. 1985. Immunotoxins. Trends Biotechnol. 3: 175–79.

    Article  CAS  Google Scholar 

  7. Boitieux, J.L., Desmet, G., Wilson, G. and Thomas, D. 1990. The specific immobilization of antibody fragments on membrane for the development of multifunctional biosensors. Ann. N. Y. Acad. Sci. 613: 390–395.

    Article  CAS  Google Scholar 

  8. North, J.R. 1985. Immunosensors:antibody-based biosensors. Trends Biotechnol. 3: 80–86.

    Article  Google Scholar 

  9. Jimbo, Y. and Saito, M. 1988. Orientation-controlled immobilization of protein molecules on thin film organic films deposited by the plasma technique. J. Mol. Electron. 4: 111–18.

    CAS  Google Scholar 

  10. Spitznagel, T.M., Jacobs, J.W. and Clark, D.S. Random and site specific immobilization of catalytic antibodies. Enz. Microb. Technol. Submitted

  11. Janda, K.D., Ashley, J.A., Jones, T.M., McLeod, D.A., Schloeder, D.M. and Weinhouse, M.I. 1990. Immobilized catalytic antibodies in aqueous and organic solvents. J. Am. Chem. Soc. 112: 8886–8888.

    Article  CAS  Google Scholar 

  12. Eveleigh, J.W. and Levy, D.E. 1977. Immunochemical characteristics and preparative application of agarose-based immunosorbents. J. Solid-Phase Biochem. 2: 45–78.

    CAS  Google Scholar 

  13. Olson, W.C., Spitznagel, T.M. and Yarmush, M.L. 1989. Dissociation kinetics of antigen-antibody interactions: studies on a panel of anti-albumin monoclonal antibodies. Mol. Immunol. 26: 129–36.

    Article  CAS  Google Scholar 

  14. Guzov, V.M. and Usanov, S.A. 1988. Optimization of antibodies against carcinoembryonic antigen on insoluble matrices. Equilibrium parameters of the interaction of immobilized antibodies with antigen. Biokhimiya. 53: 1772–1782.

    CAS  Google Scholar 

  15. Strauss, W.M., Broze, G.J., Miletich, J.P. and Null, H.R. 1987. Mass transfer in agarose-based immunoadsorbent columns. Biotechnol. Appl. Biochem. 9: 462–477.

    CAS  Google Scholar 

  16. Sada, E., Katoh, S., Sukai, K., Tohma, M. and Kondo, A. 1986. Adsorption equilibrium in immuno-affinity chromatography with polyclonal and monoclonal antibodies. Biotechnol. Bioeng. 28: 1497–1502.

    Article  CAS  Google Scholar 

  17. Eisen, H.N., Simms, E.S. and Potter, M. 1968. Mouse myeloma proteins with antihapten antibody activity. The protein produced by plasma cell tumor MOPC-315. Biochemistry 7: 4126–34.

    Article  CAS  Google Scholar 

  18. Haselkorn, D., Friedman, S., Givol, D. and Pecht, I. 1974. Kinetic mapping of the antibody combining site by chemical relaxation spectrometry. Biochemistry 13: 2210–2222.

    Article  CAS  Google Scholar 

  19. Hochmann, J., Inbar, D. and Givol, D. 1973. An active antibody fragment (Fv) composed of the variable portions of heavy and light chains. Biochemistry 12: 1130–35.

    Article  Google Scholar 

  20. Baldwin, E.P. 1989. Construction, expression, and mutagenesis of genes encoding the variable region domain of MOPC315, p. 68. Thesis, Department of Chemistry, University of California, Berkeley.

  21. Lin, J.N., Chang, I.-N., Andrade, J.D., Herron, J.N. and Christensen, D.A. 1991. Comparison of site-specific coupling chemistry for antibody immobilization on different solid supports. J. Chromatog. 542: 41–54.

    Article  CAS  Google Scholar 

  22. Lin, J.N., Andrade, J.D. and Chang, I.-N. 1989. The influence of adsorption of native and modified antibodies on their activity. J. Immunol. Meth. 125: 67–77.

    Article  CAS  Google Scholar 

  23. Sportsman, J.R. and Wilson, G.S. 1980. Chromatographic properties of silica-immobilized antibodies. Anal. Chem. 52: 2013–2018.

    Article  CAS  Google Scholar 

  24. Ernst-Cabrera, K. and Wilchek, M. 1988. High-performance affinity chromatography. Trends Anal. Chem. 7: 58–63.

    Article  CAS  Google Scholar 

  25. Parikh, I., March, S. and Cuatrecasas . 1974. Topics in the methodology of substitution reactions with agarose. Methods in Enzymol. 34: 77–102.

    Article  CAS  Google Scholar 

  26. Inbar, D., Rotman, M. and Givol, D. 1971. Crystallization with hapten of the Fab′ fragment from a mouse IgA myeloma protein with antidinitrophenyl activity. J. Biol. Chem. 246: 6272–75.

    CAS  PubMed  Google Scholar 

  27. Goetzl, E.J. and Metzger, H. 1970. Affinity labeling of a mouse myeloma protein which binds nitrophenyl ligands. Kinetics of labeling and isolation of a labeled peptide. Biochemistry 9: 1267–78.

    Article  CAS  Google Scholar 

  28. Weetall, H.H. 1976. Covalent coupling methods for inorganic support materials. Methods in Enzymol. 44: 134–148.

    Article  CAS  Google Scholar 

  29. Ternynck, T. and Avrameas, S. 1976. Polymerization and immobilization of proteins using ethylchloroformate and glutaraldehyde, p. 29–35. In: Immuno-adsorbents in protein purification. Ruolsahti, E. (Ed.). University Park Press, Baltimore, MD.

    Google Scholar 

  30. Skerker, P.S. and Clark, D.S. 1988. Catalytic properties and active-site structural features of immobilized horse liver alcohol dehydrogenase. Biotechnol. Bioeng. 32: 148–58.

    Article  CAS  Google Scholar 

  31. Hammers, G.G. 1982. Regulation of enzyme activity, p. 161–162. In: Enzyme Catalysis and Regulation. Hammes, G. G. (Ed.). Academic Press, New York.

  32. Rittenberg, M.B. and Amkraut, A.A. 1966. Immunogenicity of trinitrophenyl-hemocyanin: production of primary and secondary anti-hapten precipitins. J. Immunol. 97: 421–430.

    CAS  PubMed  Google Scholar 

  33. Little, R.J. and Eisen, H.N. 1967. Preparation of immunogenic 2,4-dini-trophenyl and 2,4,6-trinitrophenyl proteins. Methods Immunoi. Immunochem. 1: 128–133.

    Google Scholar 

  34. Gabel, D. 1973. The denaturation by urea and guanidinium chloride of trypsin and N-acetylated-trypsin derivatives bound to sephadex and agarose. Eur. J. Biochem. 33: 348–356.

    Article  CAS  Google Scholar 

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  1. Douglas S. Clark: Corresponding author.

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  1. Department of Chemical Engineering, University of California, Berkeley, CA, 94720

    Thomas M. Spitznagel & Douglas S. Clark

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Spitznagel, T., Clark, D. Surface-Density and Orientation Effects on Immobilized Antibodies and Antibody Fragments. Nat Biotechnol 11, 825–829 (1993). https://doi.org/10.1038/nbt0793-825

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