Monoclonal Antibodies

Heddy Zola, Child Health Research Institute, Adelaide, Australia

Published online: September 2010

DOI: 10.1002/9780470015902.a0001205.pub3

Full Article on Wiley Online Library

Antibodies bind other molecules strongly and specifically and are therefore useful as reagents in research, diagnosis and therapy. Antibodies taken from the blood of immunised animals are a mixture of different antibodies produced by different cells (they are described as polyclonal). Monoclonal antibodies are antibodies with a unique specificity, generally made by cloning cells containing a particular antibody gene set to produce a population of identical cells, derived from a single cell, which all produce the same antibody. Monoclonal antibodies are therefore much more specific than polyclonal antibodies. Monoclonal antibodies can be made in cell culture and are therefore also more reproducible from batch to batch than polyclonal antibodies. Monoclonal antibodies have become the preferred reagents in many research and diagnostic applications and are increasingly used in therapy of cancer and immunological disorders, generating a multi-billion dollar industry.

Key Concepts:

Antibodies form part of the mammalian immune response, binding to foreign molecules to neutralise or remove them.
By injecting an animal with a target molecule, we can make an antibody that binds the target molecules strongly and specifically.
One immune cell makes just one antibody, but from the blood, we get a mixture of the antibodies produced by many immune cells.
By isolating a single immune cell making a single antibody of interest and converting it to a dividing, growing clone of cells (cloning the cell), we can make large quantities of identical antibody – monoclonal antibody.
Monoclonal antibodies provide specific reagents for almost any molecular structure above a minimal size.
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 to remove harmful components ranging from toxins to cancer cells.
Advances in molecular biological technologies make it possible to modify monoclonal antibodies to achieve therapeutic objectives with minimal side effects.

Keywords: monoclonal antibodies; hybridoma technology; immunoassay

Further Reading
	Adams GP, Schier R, McCall AM et al. (2001) High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules. Cancer Research 61: 4750–4755.
	Al-Ejeh F, Darby JM, Tsopelas C et al. (2009) APOMAB, a La-specific monoclonal antibody, detects the apoptotic tumor response to life-prolonging and DNA-damaging chemotherapy. PLoS One 4: e4558.
	Holliger P and Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nature Biotechnology 23: 1126–1136.
	Kohler G and Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497.
	Ruszkiewicz A, Bennett G, Moore J et al. (2002) Correlation of mismatch repair genes immunohistochemistry and microsatellite instability status in HNPCC-associated tumours. Pathology 34: 541–547.
	Thiel MA, Coster DJ, Standfield SD et al. (2002) Penetration of engineered antibody fragments into the eye. Clinical and Experimental Immunology 128: 67–74.
	Thomas DA, O'Brien S and Kantarjian HM (2009) Monoclonal antibody therapy with rituximab for acute lymphoblastic leukemia. Hematology/Oncology Clinics of North America 23: 949–971.
	Winter G, Griffiths AD, Hawkins RE and Hoogenboom HR (1994) Making antibodies by phage display. Annual Review of Immunology 12: 433–455.
	Winter G and Milstein C (1991) Man-made antibodies. Nature 349: 293–299.
	book Zola H (1987) Monoclonal Antibodies: A Manual of Techniques. Boca Raton, FL: CRC Press.
	book Zola H (2000) Monoclonal Antibodies: The Basics Oxford. UK: Bios Scientific Publishers.

Article Title:	Monoclonal Antibodies
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	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 non-neoplastic tissue including lamina propria lymphocytes show MSH-2 protein expression. Courtesy of Dr Andrew Ruszkiewicz, SA Pathology, South Australia.
	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 include 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.

Monoclonal Antibodies

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