Advertisement

Measurement of Antibody Concentrations by Hemagglutination

  • Jenny Phillips
  • Geoff Hale
Part of the Methods in Molecular Medicine book series (MIMM, volume 40)

Abstract

During every stage of development and production of diagnostic or therapeutic antibodies, it is necessary to have an assay to measure antibody concentration. Several techniques are routine in virtually all antibody laboratories. High-performance liquid chromatograpy (HPLC) using an affinity matrix (protein A or protein G) is rapid and quantitative and measures most types of antibody, though the equipment is quite costly. Enzyme-linked immunosorbent assay (ELISA) is widely used and extremely versatile. By judicious choice of anti-Ig reagents, specific for heavy chain, light chains, or particular domains, it is possible to screen for almost any desired Ig molecule or fragment. This is specially useful when analyzing a complex mixture (1). Native gel electrophoresis is a useful technique for screening relatively concentrated samples (e.g., from fermentors) since different antibodies can be readily distinguished by their characteristic mobilities. However, none of these methods are really suited to the rapid semiquantitative testing of huge numbers of samples that is often necessary early in a project (when screening for a rare hybridoma or transfectant, or for somatic mutants) and for routine analysis of process samples during cell culture. Instead we have found that red cell agglutination as originally developed by Coombs (2,3), is convenient, quick, and very cheap.

Keywords

Antibody Concentration Trisodium Citrate Affinity Matrix Couple Cell Chromic Chloride 
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.

References

  1. 1.
    Clark, M. R. and Waldmann, H. (1987) T cell killing of target cells induced by hybrid antibodies: a comparison of two bispecific monoclonal antibodies. J. Natl. Cancer Inst. 79, 1393–1401.PubMedGoogle Scholar
  2. 2.
    Coombs, R. R. A., Scott, M. L., and Cranage, M. P. (1987) Assays using red-cell labelled antibodies. J. Immunol. Methods 101, 1–14.CrossRefPubMedGoogle Scholar
  3. 3.
    Clark, M. R. (1986) The detection and characterization of antigen-specific monoclonal antibodies using anti-immunoglobulin isotype antibodies coupled to red blood cells. Methods Enzymol. 121, 548–555.CrossRefPubMedGoogle Scholar
  4. 4.
    Hale, G., Cobbold, S. P., Waldmann, H., Easter, G., Matejtschuk, P., and Coombs, R. R. A. (1987) Isolation of low-frequency class-switch variants from rat hybrid myelomas. J. Immunol. Methods 103, 59–67.CrossRefPubMedGoogle Scholar
  5. 5.
    Hale, G., Drumm, A., Harrison, P., and Phillips, J. (1994) Repeated cleaning of protein A affinity column with sodium hydroxide. J. Immunol. Methods 171, 15–21.CrossRefPubMedGoogle Scholar
  6. 6.
    Cranage, M. P., Gurner, B. W., and Coombs, R. R. A. (1983) Glutaraldehyde stabilisation of antibody-linked erythrocytes for use in reverse passive and related hemagglutination assays. J. Immunol. Methods 64, 7–16.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Jenny Phillips
    • 1
  • Geoff Hale
    • 2
  1. 1.Therapeutic Antibody CentreOxfordUK
  2. 2.Sir William Dunn School of PathologyOxford UniversityOxfordUK

Personalised recommendations