Separation techniques

  • David Newman
  • Christopher Price


All heterogeneous immunoassays require a procedure to separate the bound labelled ligand from the free. There have been several reviews focussing on this important aspect of immunoassay, namely Ratcliffe in 1974 (1), a multi-author section in the book edited by Hunter and Corrie in 1983 (2) and more recently by Gosling in 1990 (3). Here we shall summarise the salient features that are expected of a separation technique, and with passing reference to historical approaches, focus on those in current use with concluding comments on some future possibilities.


Magnetic Particle Separation Technique Antibody Reaction Separation System Cyanogen Bromide 
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  1. 1.
    Ratcliffe, J.G. (1974) Separation techniques in saturation analysis. Brit. Med. Bull. 30, 32–7.PubMedGoogle Scholar
  2. 2.
    Hunter, W.M. and Corne, J.E.T. (eds.) (1983) Immunoassays for Clinical Chemistry. 2nd Edn. pp. 701, Churchill-Livingstone, Edinburgh.Google Scholar
  3. 3.
    Gosling, J.P. (1990) A decade of development in immunoassay methodology. Clin. Chem. 36, 1408–27.PubMedGoogle Scholar
  4. 4.
    Wide, L. and Porath, J. (1966) Radioimmunoassays of proteins with the use of Sephadex coupled antibodies. Biochim. Biophys. Acta. 130, 257–62.CrossRefGoogle Scholar
  5. 5.
    Miles, L.E.M. and Hales, C.N. (1968) Labelled antibodies and immunological assay systems. Nature. 219, 186–9.PubMedCrossRefGoogle Scholar
  6. 6.
    Utiger, R.D., Parker, M.L., Daughaday, W.H. (1962) Studies on human growth hormone. I A radioimmunoassay for human growth hormone. J. Clin. Invest. 41, 254–61.PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Edwards, R. (1983) The development and use of a PEG assisted second-antibody as a separation technique in RIA. In: Immunoassays for Clinical Chemistry. (eds. Hunter, W.M. and Corrie, J.E.T.) 2nd Edn, p 139–46, Churchill-Livingstone, Edinburgh.Google Scholar
  8. 8.
    Oliver, J.R., Hakendorf, P., Zeegers, P., Ross, W. (1982) A proposed simple method for detection and measurement of antibodies to insulin in serum by use of staphylococcus aureus containing protein A. Clin. Chem. 28, 121–3.PubMedGoogle Scholar
  9. 9.
    Newman, D.J., Medcalf, E.A., Gorman, E.G., Price, C.P. (1989) A novel solid phase enzyme-immunoassay for beta-2-microglobulin. Biologie Prospective. Comptes rendus de 7e Colloque de Ponte-aMousson. (eds. Galteau, M-M., Siest, G., Henny, J.) p 119–22, John Libbey, Eurotext.Google Scholar
  10. 10.
    Catt, K. and Tregear, C.W. (1967) Solid-phase radioimmunoassay in antibody coated tubes. Science. 158, 1570–2.PubMedCrossRefGoogle Scholar
  11. 11.
    Donini, S. and Donini, P. (1969) Radioimmunoassay employing polymerised antisera. Acta. Endocrinol. Suppl. 142, 25–8.Google Scholar
  12. 12.
    Bolton, A.E. and Hunter, W.M. (1973) The use of antisera covalently coupled to agarose, cellulose and Sephadex in radioimmunoassays for proteins and haptens. Biochim. Biophys. Acta. 329, 318–30.PubMedCrossRefGoogle Scholar
  13. 13.
    Axon, R., Porath, J., Ernback, S. (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen bromide. Nature. 214, 1302–4.CrossRefGoogle Scholar
  14. 14.
    Chapman, R.S., Sutherland, R.M., Ratcliffe, J.G. (1983) Application of 1,1’-carbonyldiimidazole as a rapid practical method for the production of solid-phase immunoassay reagents. In: Immunoassays for Clinical Chemistry. (eds. Hunter, W.M. and Corrie, J.E.T.) 2nd Edn, p 178–90, Churchill-Livingstone, Edinburgh.Google Scholar
  15. 15.
    Wood, W.G., and Gadow, A. (1983) Immobilisation of antibodies and antigens on macro solid phases: a comparison between adsorptive and covalent binding. Part 1 of a critical study of macro solid phases for use in immunoassay systems. J. Clin. Chem. Clin. Biochem. 21, 789–97.PubMedGoogle Scholar
  16. 16.
    Kemeny, D.M. and Challacombe, S.J. (1988) Microtitre plates and other solid-phase supports. In: ELISA and other Solid Phase Immunoassays: Theoretical and Practical Aspects. (eds. Kemeny, D.J. and Challacombe, S.J. ) p 31–56, John Wiley and Sons, Chichester.Google Scholar
  17. 17.
    Thakkar, H., Davey, C.L., Medcalf, E.A., et al. (1991) Stabilisation of turbidimetric immunoassay by covalent coupling of antibody to latex particles. Clin. Chem., in press.Google Scholar
  18. 18.
    Voller, A., Bidwell, D.E., Bartlett, A. (1979) The Enzyme-linked Immunosorbent Assay (ELISA). Dynatech Europe, UK.Google Scholar
  19. 19.
    Engvall, E. and Perlmann, P. (1971) Enzyme-linked immunosorbent assay (ELISA): quantitative assay of IgG. Immunochemistry. 8, 871.PubMedCrossRefGoogle Scholar
  20. 20.
    Nygren, H. and Stenberg, A.M. (1989) Immunochemistry at interfaces. Immunology. 66, 321–7.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Stenberg, M. and Nygren, H. (1988) Kinetics of antigenantibody reactions at solid-liquid interfaces. J. Immunol. Meth. 113, 3–15.CrossRefGoogle Scholar
  22. 22.
    Engbaek, F., Christensen, S.E., Jespersen, B. (1989) Enzyme immunoassay of haemoglobin Al,: analytical characteristics and clinical performance for patients with diabetes mellitus, with and without uremia. Clin. Chem. 35, 93–7.PubMedGoogle Scholar
  23. 23.
    Barnard, G. and Kohen, F. (1990). Idiometric assay: non competitive immunoassay for small molecules typified by the measurement of estradiol in serum. Clin. Chem. 36, 1945–50.PubMedGoogle Scholar
  24. 24.
    Wood, W. (1991) Matrix effects in immunoassays. Scand. J. Clin. Lab. Invest. 51, Suppl. 205, 105–12.CrossRefGoogle Scholar
  25. 25.
    Ehrhardt, V., Neumeier, D., Meyer, H.D. (1988) Mechanization of heterogeneous immunoassays. J. Clin. Immunoassay. 11, 74–80.Google Scholar
  26. 26.
    Wright, J.F. and Hunter, W.M. (1983) The sucrose layering separation: A non-centrifugation system. In: Immunoassays for Clinical Chemistry. (eds. Hunter, W.M. and Corrie, J.E.T.) 2nd Edn, p 170–7, Churchill-Livingstone, Edinburgh.Google Scholar
  27. 27.
    Fiore, M., Mitchell, J., Doan, T., et al. (1988) The Abbott ImXTM automated benchtop immunochemistry analyzer system. Clin. Chem. 34, 1726–32.PubMedGoogle Scholar
  28. 28.
    Freytag, J.W., Dickinson, J.C., Tseng, S.Y. (1984) A high sensitivity affinty-column-mediated immunometric assay as exemplified by digoxin. Clin. Chem. 30, 417–20.PubMedGoogle Scholar
  29. 29.
    Mériadec, B., Jolu, J-P., Henry, R. (1979) A new and universal separation system technique for the `Centria’ automated radioimmunoassay system. Clin. Chem. 25, 1596–9.PubMedGoogle Scholar
  30. 30.
    Forrest, G.C. and Rattle, S.J. Magnetic particle radioimmunoassay. In: Immunoassays for Clinical Chemistry. (eds. Hunter, W.M. and Corrie, J.E.T.) 2nd Edn, p 147–62, Churchill-Livingstone, Edinburgh.Google Scholar
  31. 31.
    Hersh, L.S. and Yaverbaum, S. (1975) Magnetic solid phase radioimmunoassay. Clin. Chim. Acta. 63, 69–72.Google Scholar
  32. 32.
    Robinson, P.J., Dunhill, P., Lilly, M.D. (1973) The properties of magnetic supports in relation to immobilized enzyme reagents. Biotech. Bioeng. 15, 603–6.CrossRefGoogle Scholar
  33. 33.
    Forrest, G.C. (1977) Development and application of a fully automated continuous flow radioimmunoassay system. Ann. Clin. Biochem. 14, 1–11.PubMedCrossRefGoogle Scholar
  34. 34.
    Nargessi, R.D., Landon, J., Pourfarzaneh, M., Smith, D.S. (1978) Solid phase fluoroimmunoassay of human albumin in biological fluids. Clin. Chim. Acta. 89, 455–60PubMedCrossRefGoogle Scholar
  35. 35.
    Harmer, I.J. and Samuel, D. (1989) The FITC-anti-FITC system is a sensitive alternative to biotin-streptavidin in ELISA. J. Immunol. Method. 122, 115–21.CrossRefGoogle Scholar
  36. 36.
    Milne, C.N., Pritchard, G.J., Allen, G.J., et al. (1988) Automation of enzyme immunoassays for hormones. J. Endocr. 119, Suppl. Abstract, 89.Google Scholar
  37. 37.
    Al-Dujaili, E.A.S., Forrest, G.C., Edwards, C.R.W., Landon, J. (1979) Evaluation and application of magnetizable charcoal for separation in radioimmunoassays. Clin. Chem. 25, 1402–5.PubMedGoogle Scholar
  38. 38.
    Birkmeyer, R.C., Diaco, R., Hutson, D.K., et al. (1987) Application of novel chromium dioxide magnetic particles to immunoassay development. Clin. Chem. 33, 1543–7.PubMedGoogle Scholar
  39. 39.
    Hansen, J.B., Lay, H.P., Janes, C.J., et al. (1990) A rapid and specific assay for the du Pont aca discrete clinical analyser, performed directly on whole blood. Transpl. Proc. 22, 1189–92.Google Scholar
  40. 40.
    Giegel, J.L., Brotherton, M.M., Cronin, P., et al. (1982) Radial partition immunoassay. Clin. Chem. 28, 1894–8.PubMedGoogle Scholar
  41. 41.
    Valkirs, G.E. and Barton, R. (1985) Immuno ConcentrationTM — a new format for solid-phase immuno assays. Clin. Chem. 31, 1427–31PubMedGoogle Scholar
  42. 42.
    Unipath Clear Blue pregnancy test, Unipath-Oxoid, Bedford, UK.Google Scholar
  43. 43.
    Wang, R., Merrill, B., Maggio, E.T. (1980) A simplified solid phase immunofluorescence assay for measurement of serum immunoglobulins. Clin. Chim.Acta. 102, 169–77.PubMedCrossRefGoogle Scholar
  44. 44.
    Zuk, R. F., Ginsberg, V. K., Houts, T., (1985) Enzyme immunochromatography a quantitative immunoassay requiring no instrumentation. Clin. Chem. 31, 1144–50.PubMedGoogle Scholar
  45. 45.
    Pappas, M.G. (1988) Dot enzyme-linked immunosorbent assays. In: Complementary Immunoassays. (ed Collins, W.P. ) p 113–34, John Wiley and Sons., Chichester.Google Scholar
  46. 46.
    Chan, D.W., Waldron, C., Bill, M.J., Drew, H. (1987) The performance of a totally automated enzyme immunoassay system (ES 600). Clin. Chem. 33, 947. [Abstract].Google Scholar
  47. 47.
    Rasmussen, S.E. (1988) Solid phases and chemistries. In: Complementary Immunoassays. (ed Collins, W.P. ) p 43–55, John Wiley and Sons., Chichester.Google Scholar
  48. 48.
    Clark, P.M.S., and Price, C.P. (1986) Enzyme amplified immunoassays: a new ultrasensitive assay of thyrotropin evaluated. Clin. Chem. 32, 88–92.PubMedGoogle Scholar
  49. 49.
    Hiratsuka, N., Mihara, Y. and Miyazako, T. (1982) Method for immunological assay using multilayer analysis sheet. U. S. Patent No. 4,337,065.Google Scholar

Copyright information

© Palgrave Macmillan, a division of Macmillan Publishers Limited 1991

Authors and Affiliations

  • David Newman
  • Christopher Price

There are no affiliations available

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