Monoclonal Antibodies

Abstract

Antibodies bind to target molecules strongly and specifically and are therefore useful reagents in research, diagnosis and therapy. Antibodies taken from the blood of immunised animals are a mixture of different immunoglobulins produced by many B cells, and are known as polyclonal antibodies. In contrast, monoclonal antibodies are antibodies with a unique specificity, generally made by cells containing a particular immunoglobulin gene set derived from a single cell or a clone of B cells. Monoclonal antibodies can be produced in cell culture and are therefore more reproducible from batch to batch than polyclonal antibodies. Most of the monoclonal antibodies currently used were generated from mice using the hybridoma technology. However, recently powerful alternative technologies have been developed for the generation of monoclonal antibodies, especially fully human antibodies. These methods are based on the cloning and sequencing of the immunoglobulin genes from antigen‐specific B cells followed by the generation of recombinant antibodies. Monoclonal antibodies have become essential reagents in many research and diagnostic applications, and are increasingly used in therapy, generating a multibillion dollar industry.

Key Concepts

  • Antibodies form part of the vertebrate immune response, binding with high affinity and specificity to target molecules (antigens).
  • Antibodies are able to neutralise and remove antigens and modulate biological functions.
  • Antibodies that bind to target molecules can be made by injecting an animal with antigens, from immune individuals or synthetic libraries.
  • One B cells makes just one antibody with a unique specificity, but from the blood, we get a mixture of the antibodies produced by many B cells (polyclonal antibodies).
  • A monoclonal antibody is an antibody, which is obtained by expanding and immortalising a single clone of B‐cells with a defined specificity.
  • Monoclonal antibodies provide specific reagents for almost any molecular structure.
  • Monoclonal antibodies can be used to identify, quantify, isolate or remove the target molecule in complex biological mixtures or in tissues.
  • Monoclonal antibodies can be injected into patients for the treatment of a wide range of diseases including infections, cancer, cardiovascular diseases and autoimmune diseases.
  • Advances in molecular biological technologies make it possible to modify monoclonal antibodies to achieve enhanced therapeutic effects with minimal side effects.

Keywords: monoclonal antibodies; hybridoma technology; immunoassay; recombinant antibodies; immunotherapy

Figure 1. Schematic representation of the process of immortalising an antibody‐producing clone by hybridisation, cloning and selection of clones producing the desired antibodies. Notes: Ab, antibody and Ag, antigen.
Figure 2. Examples of the use of monoclonal antibodies to identify particular molecules and cell types in tissues. (a) Staining of human lymph node tissue with a monoclonal antibody against a protein called CD19 and fluorescence microscopy. The protein is expressed on the surface of B lymphocytes, which are responsible for antibody production. The egg‐shaped structure is a follicle of B lymphocytes, while the surrounding area (unstained) contains principally T lymphocytes, with a sprinkling of B lymphocytes. The follicle contains a germinal centre. Germinal centres develop in response to infection or other antigenic challenge. In the germinal centre, B lymphocytes divide rapidly, and the genes coding for the antibody made by the cell are mutated and selected to give strong binding to antigen. (b) Staining of breast cancer tissue section with two antibodies against different molecules, conjugated to different enzymes and detected using different colour‐producing (chromogenic) enzyme substrates. Red staining is for the epithelial tissue marker AE1/3 and brown staining for the breast cancer marker Erb‐b2. Courtesy of Dr Andrew Ruszkiewicz, SA Pathology, South Australia. (c) Staining of colonic cancer tissue section with antibody against MSH‐2, a protein involved in DNA mismatch repair, showing abnormal expression. The neoplastic glands show loss of nuclear staining, while nonneoplastic tissue including lamina propria lymphocytes show MSH‐2 protein expression. Courtesy of Dr Andrew Ruszkiewicz.
Figure 3. The analytical power of a panel of monoclonal antibodies against molecules on the surface of blood cells. (a) Light scatter distribution of blood cells analysed in a flow cytometer, allowing the selection of the lymphocyte population for further analysis. If monoclonal antibodies against a T‐lymphocyte marker and a B‐lymphocyte marker, each attached to a different fluorescent dye, are added to the blood sample, the pattern seen in (b) can be obtained, allowing the selection of T cell for further analysis. (c) Resolution of T lymphocytes into two populations: lymphocytes marked with a monoclonal antibody against CD4, identifying a population containing ‘helper’ cells, which provide positive signals in an immune response, and the CD8 population, which includes suppressive activity. (d) If the CD4 lymphocytes are selected for further analysis, they may be further separated into cells (CD45RO‐positive) that have previously been activated (memory cells) and those that have not previously been activated (naïve cells). (e) These can be subdivided in turn according to the cytokines they make – cells that make IL‐4 tend to favour antibody responses, while cells that make IL‐2 tend to stimulate cell‐mediated immunity. (Note that panels (a)–(d) show actual data, while panel (e) shows simulated data.) Technical limitations in flow cytometry instrumentation and the number of different fluorescent dyes available place limits on the extent of the analysis. Widely available instruments can analyse on the basis of three colours simultaneously (allowing, e.g. the identification or separation of CD4 cells for further analysis), while more sophisticated research methods allow up to 11 simultaneous antibody markers, more than adequate to conduct the entire series shown in the figure. In practice, some steps can be left out; there is no need, for example, to include a B‐cell marker or CD8 or CD45RA in identifying the IL‐4‐secreting helper T cells.
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Further Reading

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Zola, Heddy, and Engel, Pablo(Oct 2015) Monoclonal Antibodies. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0001205.pub4]