L V

Screen supernatants for each clone of anti-X antibody and expand positive clones

FIGURE 5-9 The generation of monoclonal antibodies. In this procedure, spleen cells from a mouse that has been immunized with a known antigen or mixture of antigens are fused with an enzyme-deficient partner myeloma cell line, with use of chemicals such as polyethylene glycol that can facilitate the fusion of plasma membranes and the formation of hybrid cells that retain many chromosomes from both fusion partners. The myeloma partner used is one that does not secrete its own Igs. These hybrid cells are then placed in a selection medium that permits the survival of only immortalized hybrids; these hybrid cells are then grown as single cell clones and tested for the secretion of the antibody of interest. The selection medium includes hypoxanthine, aminopterin, and thymidine and is therefore called HAT medium. There are two pathways of purine synthesis in most cells, a de novo pathway that needs tetrahydrofolate and a salvage pathway that uses the enzyme hypoxanthine-guanine phosphoribosyl-transferase (HGPRT). Myeloma cells that lack HGPRT are used as fusion partners, and they normally survive using de novo purine synthesis. In the presence of aminopterin, tetrahydrofolate is not made, resulting in a defect in de novo purine synthesis and also a specific defect in pyrimidine biosynthesis, namely, in generating TMP from dUMP. Hybrid cells receive HGPRT from the splenocytes and have the capacity for uncontrolled proliferation from the myeloma partner; if they are given hypoxanthine and thymidine, these cells can make DNA in the absence of tetrahydrofolate. As a result, only hybrid cells survive in HAT medium.

FIGURE 5-9 The generation of monoclonal antibodies. In this procedure, spleen cells from a mouse that has been immunized with a known antigen or mixture of antigens are fused with an enzyme-deficient partner myeloma cell line, with use of chemicals such as polyethylene glycol that can facilitate the fusion of plasma membranes and the formation of hybrid cells that retain many chromosomes from both fusion partners. The myeloma partner used is one that does not secrete its own Igs. These hybrid cells are then placed in a selection medium that permits the survival of only immortalized hybrids; these hybrid cells are then grown as single cell clones and tested for the secretion of the antibody of interest. The selection medium includes hypoxanthine, aminopterin, and thymidine and is therefore called HAT medium. There are two pathways of purine synthesis in most cells, a de novo pathway that needs tetrahydrofolate and a salvage pathway that uses the enzyme hypoxanthine-guanine phosphoribosyl-transferase (HGPRT). Myeloma cells that lack HGPRT are used as fusion partners, and they normally survive using de novo purine synthesis. In the presence of aminopterin, tetrahydrofolate is not made, resulting in a defect in de novo purine synthesis and also a specific defect in pyrimidine biosynthesis, namely, in generating TMP from dUMP. Hybrid cells receive HGPRT from the splenocytes and have the capacity for uncontrolled proliferation from the myeloma partner; if they are given hypoxanthine and thymidine, these cells can make DNA in the absence of tetrahydrofolate. As a result, only hybrid cells survive in HAT medium.

SYNTHESIS, ASSEMBLY, AND EXPRESSION OF Ig MOLECULES 99

TABLE 5-3 Monoclonal Antibodies of Therapeutic Significance

Target

Effect

Diseases

Was this article helpful?

0 0
How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment