Starting in the 1950s, biochemists in search of a homogeneous preparation of antibody that they could subject to detailed chemical analysis turned to proteins produced by patients with multiple myeloma, a common tumor of plasma cells. It was known that antibodies are normally produced by plasma cells, and because this disease is associated with the presence of large amounts of a homogeneous gamma globulin called a myeloma protein in the patient’s serum, it seemed likely that myeloma proteins would serve as models for
Fig. A.8 The Coombs direct and indirect anti-globulin tests for antibody against red blood cell antigens. An Rh– mother of an Rh+ fetus can become immunized to fetal red blood cells that enter the maternal circulation at the time of delivery. In a subsequent pregnancy with an Rh+ fetus, IgG anti-Rh antibodies can cross the placenta and damage the fetal red blood cells. In contrast to anti-Rh antibodies, maternal anti-ABO antibodies are of the IgM isotype and cannot cross the placenta, and so do not cause harm. Anti-Rh antibodies do not agglutinate red blood cells, but their presence on the fetal red blood cell surface can be shown by washing away unbound immunoglobulin and then adding antibody against human immunoglobulin, which agglutinates the antibody-coated cells. Anti-Rh antibodies can be detected in the mother’s serum in an indirect Coombs test; the serum is incubated with Rh+ red blood cells, and once the antibody has bound, the red blood cells are treated as in the direct Coombs test.
Fig. A.9 The production of monoclonal antibodies. Mice are immunized with antigen A and given an intravenous booster immunization 3 days before they are killed, in order to produce a large population of spleen cells secreting specific antibody. Spleen cells die after a few days in culture. To produce a continuous source of antibody they are fused with immortal myeloma cells by using polyethylene glycol (PEG) to produce a hybrid cell line called a hybridoma. The myeloma cells are selected beforehand to ensure that they are not secreting antibody themselves and that they lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT); without this enzyme, unfused myeloma cells are sensitive to the hypoxanthine–aminopterin–thymidine (HAT) medium, which is used to select hybrid cells. The HGPRT gene contributed by the spleen cell allows hybrid cells to survive in the HAT medium, and only hybrid cells can grow continuously in culture, because of the malignant potential contributed by the myeloma cells combined with the finite life-span of unfused spleen cells. Unfused myeloma cells and unfused spleen cells therefore die in the HAT medium, as shown here by cells with dark, irregular nuclei. Individual hybridomas are obtained by single cell dilution and then screened for antibody production, and single clones that make antibody of the desired specificity can be isolated and grown. The cloned hybridoma cells are grown in bulk culture to produce large amounts of antibody. As each hybridoma is descended from a single cell, all the cells of a hybridoma cell line make the same antibody molecule, which is thus called a monoclonal antibody.
normal antibody molecules. Thus, much of the early knowledge of antibody structure came from studies on myeloma proteins. These studies showed that monoclonal antibodies could be obtained from immortalized plasma cells. However, the antigen specificity of most myeloma proteins was unknown, which limited their usefulness as objects of study or as immunological tools.