Monoclonal vs Polyclonal antibodies

Polyclonal Antibodies

Although each B-cell clone produces an antibody which is specific for one particular epitope of an antigen, the serum antibodies produced in response to a complex antigen contain a mixture of antibodies because the multiple epitopes on an antigen will induce proliferation and differentiation of a variety of B cell clones. Each one of these cell clones produce antibodies which are specific for one of the various epitopes on the antigen. This polyclonal response is advantageous for the immune system because is helps localization, phagocytosis, and complement mediated lysis of the antigen.

Monoclonal Antibodies

While polyclonal antibodies are advantageous for the immune system, the production of monoclonal antibody, which is derived from a single plasma cell and is specific for one epitope is more useful for the growing market of in vitro and in vivo diagnostic products and therapeutics. Some of the uses of monoclonal antibodies include:

• Purification of proteins. For example, one can take a complex mixture of proteins, immunize mice and then produce monoclonal antibody to the protein of interest. One can then attach this monoclonal antibody to beads to form an immunoadsorbent column which can be used to purify the protein of interest.
• Identification and isolation of various lymphocyte subpopulations. For example, monoclonal antibodies to CD4 and CD8 can be labeled with two different fluorochromes and incubated with a lymphoctye preparation. The T_H and T_c cells can then be separated in a fluorescence activated cell sorter.
• Tumor detection and elimination. Monoclonal antibodies specific for certain tumor associated membrane proteins can be produced. Tumor specific monoclonal antibody can also be conjugated to a lethal toxin to form what is sometimes called an immunotxin that is capable of killing tumor cells. Such immunotoxins can be directed against cell membrane antigens of various cancers.
• Diagnostic reagents for things like bacterial and viral pathogens. Many of these test kits use strips of paper impregnated with an appropriate monoclonal antibody.

Monoclonal and polyclonal antibodies have been shown to react with surface components of cells infected by HIV primary isolates, indicating that targeted cells express surface antigens which can induce and bind antibodies.

Monoclonal Antibody Production

Kohler and Milstein (1975) were the first to demonstrate that somatic cells could be fused with murine myelomas and that monoclonal antibodies with unique specificities could be produced. A vast array of monoclonal antibodies have now been produced. In the generation of most monoclonal antibodies, mice are immunized against a specific antigen, and their cells are fused with the mouse myeloma cell.

The procedure is as follows: (1) spleen cells from immunized mice are fused, using polyehtylene glycol, with myeloma cells which are rendered drug sensitive by a mutation in a growth essential gene HGPRT (hypoxanthin-guanine-phosphoribosyltransferase). (2) the cell mixture is then cultured in medium containing the selective drug. The immune cells, although not sensitive to HGPRT, survive for only about one week in culture. The myeloma cells are drug senstivie and die within a week. The only cells that survive are those hybrid myeloma cells that obtained a normal HGPRT gene from the immune cells. These hybridomas can grow continuosly in vitro and some secrete antibody. (3) Using appropriate screening technology, clones of cells that secrete antibody of interest can be identified and expanded in vitro or in vivo to obtain large quantities of monoclonal antibody that can subsequently be purified to homogeneity.

A major obstacle in the clinical use of monoclonal antibodies in humans is that such antibodies are usually mouse antibodies and thus recognized as foreign, inducing an anti-isotype response. Thus efforts are underway to try and produce human monoclonal antibodies. One approach is to incorporate genes encoding human antibody within mice. Scid-human mice, for example, contain human B and T cells and following immunization of such mice, activated human B cells can be isolated and used to produce human monoclonal antibodies. Another approach is to engineer monoclonal antibodies using recombinant DNA technology. These vectors are then transfected into Ab lacking myeloma cells. For example, one can clone recombinant DNA containing the promoter, leader, and variable region sequences from a mouse antibody gene and the constant region exons from a human antibody gene. The antigenic specificity of this mouse-human chimera is derived from the mouse DNA whereas its isotype which is determined by the constant region is derived from the human DNA. Since the constant regions are encoded by human genes, these antibodies have fewer mouse antigenic determinants and are far less immunogenic than mouse monoclonal antibodies when administered to humans.