Enzyme-linked immunosorbent assays: These assays (known as ELISA) are performed in the wells of a microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an Ab specific for the protein (such as troponin, see p. 66) to be measured. The Ab is covalently bound to an enzyme, which will produce a colored product when exposed to its substrate. The amount of color produced is proportional to the amount of Ab present and, indirectly, to the amount of protein in a test sample.
2.
Western blots: Western blots (also called immunoblots) are similar to Southern blots, except that it is protein molecules in the sample that are separated by electrophoresis and blotted (transferred) to a membrane. The probe is a labeled Ab, which produces a band at the location of its antigen.
3.
Detecting exposure to human immunodeficiency virus: ELISA and western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV Ab present in a patient’s blood sample. ELISA are used as the primary screening tool because they are very sensitive. Because these assays sometimes give false positives, however, western blots, which are more specific, are often used as a confirmatory test (Fig. 34.24). [Note: ELISA and western blots can only detect HIV exposure after anti-HIV Ab appear in the bloodstream. PCR based testing for HIV is more useful in the first few months after exposure.]
C. Proteomics
The study of the proteome, or all the proteins expressed by a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, is known as proteomics. The 20,000–25,000 protein-coding genes of the human genome translate into well over 100,000 proteins when posttranscriptional and posttranslational modifications are considered. Although a genome remains essentially unchanged, the amounts and types of proteins in any particular cell change dramatically as genes are turned on and off. [Note: Proteomics (and genomics) required the parallel development of bioinformatics, the computer-based organization, storage, and analysis of biologic data.] Figure 34.25 compares some of the analytic techniques discussed in this chapter.
IX. GENE THERAPY
The goal of gene therapy is to treat disease through delivery of the normal, cloned DNA for a gene into the somatic cells of a patient who has a defect in that gene as a result of a disease-causing mutation. Because somatic gene therapy changes only the targeted somatic cells, the change is not passed on to the next generation. [Note: In germline gene therapy, the germ cells are modified, and so the change is passed on. A long-standing moratorium on germline gene therapy is in effect worldwide.] There are two types of gene transfer: 1) ex vivo, in which cells from the patient are removed, transduced, and returned, and 2) in vivo, in which the cells are directly transduced. Both types require use of a viral vector to deliver the DNA. Challenges of gene therapy include development of vectors, achievement of long-lived expression, and prevention of side effects such as an immune response. The first successful gene therapy involved two patients with severe combined immunodeficiency disease (SCID) caused by mutations to the gene for adenosine deaminase (ADA, see p. 301). It utilized mature T lymphocytes transduced ex vivo with a retroviral vector (Fig. 34.26). [Note: Human ADA cDNA is now used.] Since 1990, only a small number of patients (with a variety of disorders, such as hemophilia, cancers, and certain types of blindness) have been treated with gene therapy, with varying degrees of success.