Monoclonal antibodies are purified antibodies cloned from a single cell. These antibodies exhibit exceptional purity and specificity and are able to recognize and bind to a specific antigen.

Discovery of the Technique

 In 1975, Köhler, Milstein, and Jerne discovered how to fuse lymphocytes to produce a cell line that was both immortal and a producer of specific antibodies. These scientists were awarded the Nobel Prize in Physiology and Medicine in 1984 for devel oping this hybridoma (cell hybrid) from different lines of cultured myeloma cells (plasma cells derived from malignant tumor strains). To induce the cells to fuse, they used Sendai virus, an influenza virus that characteristically causes cell fusion. Initially, the scientists immunized donors with sheep erythrocytes to provide a marker for the normal cells. The hybrids were tested to determine whether they still produced antibodies against the sheep erythrocytes. Köhler discovered that some of the hybrids were manufacturing large quantities of specific anti–sheep erythrocyte antibodies.

Hybrid cells secrete the antibody that is characteristic of the parent cell (e.g., anti–sheep erythrocyte antibodies). The multi plying hybrid cell culture is a hybridoma. Hybridoma cells can be cloned. The immunoglobulins derived from a single clone of cells are termed monoclonal antibodies (MAbs).

Monoclonal Antibody Production

 Modern methods for producing MAbs are refinements of the original technique. Basically, the hybridoma technique enables scientists to inoculate crude antigen mixtures into mice and then select clones producing specific antibodies against a single cell surface antigen (Fig. 1). The process of producing MAbs takes 3 to 6 months.

Fig1. Production of monoclonal anti body (MAb). (Adapted from Forbes BA, Sahm DF, Weissfeld AS: Bailey & Scott’s diagnostic microbiol ogy, ed 12, St Louis, 2007, Mosby.)

Mice are immunized with a specific antigen; several doses are given to ensure a vigorous immune response. After 2 to 4 days, spleen cells are mixed with cultured mouse myeloma cells. Myeloma parent cells that lack the enzyme, hypoxanthine phosphoribosyl transferase, are selected. Mouse myeloma cell lines usually do not secrete immunoglobulins, thus simplifying the purification process.

Polyethylene glycol (PEG) rather than Sendai virus is added to the cell mixture to promote cell membrane fusion. Only 1 in 200,000 spleen cells actually forms a viable hybrid with a myeloma cell. Normal spleen cells do not survive in culture. The fused cell mixture is placed in a medium containing hypo xanthine, aminopterin, and thymidine (HAT medium). Aminopterin is a drug that prevents myeloma cells from making their own purines and pyrimidines; they cannot use hypoxanthine from the medium, so they die.

Hybrids resulting from the fusion of spleen cells and myeloma cells contain transferase provided by the normal spleen cells. Consequently, the hybridoma cells are able to use the hypoxanthine and thymidine in the culture medium and survive. They divide rapidly in HAT medium, doubling in number every 24 to 48 hours. About 300 to 500 hybrids can be generated from the cells of a single mouse spleen, although not all will be making the desired antibodies. After the hybridomas have been growing for 2 to 4 weeks, the supernatant is tested for specific antibody using methods such as ELISA. Clones that produce the desired antibody are grown in mass culture and recloned to eliminate non–antibody-producing cells.

Antibody-producing clones lose their ability to synthesize or secrete antibody after being cultured for several months. Hybridoma cells usually are frozen and stored in small aliquots. The cells may then be grown in mass culture or injected intra peritoneally into mice. Because hybridomas are tumor cells, they grow rapidly and induce the effusion of large quantities of fluid into the peritoneal cavity. This ascites fluid is rich in MAbs and can be easily harvested.

Uses of Monoclonal Antibodies

The greatest impact of MAbs in immunology has been on the analysis of cell membrane antigens. Because they have a single specificity rather than the range of antibody molecules present in the serum, MAbs have multiple clinical applications, including the following:

 • Identifying and quantifying hormones

• Typing tissue and blood

• Identifying infectious agents

 • Identifying clusters of differentiation for the classification of leukemias and lymphomas and follow-up therapy

• Identifying tumor antigens and autoantibodies

 • Delivering immunotherapy.

مواضيع ذات صلة

Molecular Basis of Antigen - Antibody Reactions

Role of Infections and Other Environmental Influences in Autoimmunity

Genetic Factors of Autoimmunity

الحساسية الدوائية نحو العوامل المخصرة للوصل العَصَبِي العَضَلِي

آليات حساسية السلفوناميدات ومظاهرها

Antigen - Antibody Interaction: Specificity and Cross - Reactivity

ANTIBODY SYNTHESIS

Tolerance to Commensal Microbes and Fetal Antigens

B Lymphocyte Tolerance

Peripheral T Lymphocyte Tolerance

Immune Memory

B Cells