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Mol Biotech Ch9

Mol Biotech Ch9 - Chapter 9 Molecular Diagnostics Molecular...

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Chapter 9: Molecular Diagnostics Molecular Biotechnology Fig. 9.1: The purpose of ELISA (enzyme linked immunosorbent assay) is to detect a particular target antigen with an antibody in a 96-well microtiter plate format. An example application would be detecting viruses in a patient’s blood serum. Add a different sample to each well. Remember to include negative and positive controls. The protein binds to the plastic after a few hours of incubation. Then rinse out the wells with buffered detergent and replace with primary antibody, which is raised in rabbits injected with target protein. Bind and rinse as before. Add the secondary antibody, which is the IgG fraction from goats injected with heterogeneous IgG fraction from rabbit. This is called goat anti-rabbit IgG. This secondary antibody is conjugated to an enzyme (e.g., horseradish peroxidase, HRP) that converts an uncolored substrate to a colored product. The amount of color produced indicates the amount of antigen in the sample. Note that the use of an enzyme allows for amplification of the signal, since it can convert many substrate molecules to a colored product. Fig. 9.2: There are multiple epitopes on most antigens . Thus, injecting a bunny with a purified protein produces a polyclonal antibody fraction in the bunny serum. Polyclonal Abs are variable in responding to particular epitopes and require a large supply of bunnies . The use of monoclonal antibodies (MAbs) obviates these problems. Fig. 9.3: How to make MAbs. B-cells make Abs. There’s a B-cell type that makes a specific Ab for each possible antigen in the universe. So if you could culture a single B- cell, you’d have a MAb cell line. B-cells don’t multiply in cell culture unless they are cancerous (“myeloma” cells). So take a culture of myeloma cells which are carefully selected to not produce Abs, then fuse them (using PEG) with spleen cells from a mouse immunized with your target protein (the spleen is rich in B-cells). The “hybridoma” cells that result are “immortal” like the parent myeloma, but produce the one specific MAb like the parent B-cell. To eliminate parent myeloma cells from the hybrid culture, use defective mutant myeloma cells that are lacking the enzyme, “HGPRT”, which normally converts hypoxanthine to guanine and adenine (purines).
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