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blood at minute concentrations, usually in the nanomo-lar range, which further complicates the problem of their detection. Consequently, the earliest methods developed for measuring hormones were bioassays that depended on the ability of a hormone to produce a characteristic biologic response. For example, induction of ovulation in the rabbit in response to an injection of urine from a pregnant woman is an indication of the presence of the placental hormone chorionic gonadotropin and is the basis for the rabbit test that was used for many years as an indicator of early pregnancy. Before hormones were identified chemically, they were quantitated in units of the biologic responses they produced. For example, a unit of insulin is defined as one-third of the amount needed to lower blood sugar in a 2-kg rabbit to convulsive levels within 3 hr. Although bioassays are now seldom used, some hormones, including insulin, are still standardized in terms of biologic units.

Immunoassays

As knowledge of hormone structure increased, it became evident that peptide hormones were not identical in all species. Small differences in amino acid sequence, which may not affect the biologic activity of a hormone, were found to produce antibody reactions after prolonged administration. This finding led to the realization that antibody-antigen reactions might be applied to the measurement of hormones in biologic fluids. Antibodies can recognize and bind with high avidity to tiny amounts of the material (antigens) that evoked their production, even in the presence of large amounts of other substances that may be similar or different. Reaction of a hormone with an antibody results in a complex with altered physical properties as compared with either free hormone or free antibody. Ingenious techniques have been devised to isolate, detect, and quantify hormone antibody complexes. Although antibodies are commonly produced with protein hormones as antigens, production of specific antibodies to nonprotein hormones can also be induced by first attaching these compounds to some protein, for example, serum albumin.

Radioimmunoassays

The earliest and still widely used application of antibodies to the measurement of hormone concentrations in biologic fluids is the radioimmunoassay. Radioimmunoassays depend on competition between a radioactively labeled hormone prepared in the laboratory and unlabeled hormone present in a biologic sample for binding to a limited amount of antibody. The sensitivity needed for detection and quantitation of tiny amounts of hormone is obtained by incorporating iodine of high specific radioactivity into tyrosine residues of peptides. For hormones that lack a site capable of incorporating iodine such as the steroids, radioactive carbon or tritium can be used or a chemical tail containing tyrosine can be added. To perform a radioimmunoassay, a sample of plasma containing an unknown amount of hormone is mixed in a test tube with a small amount of antibody and a known amount of radioactive hormone.

The unlabeled hormone in the biological sample competes with the labeled hormone for a limited number of antibody binding sites. The more hormone present in the plasma sample, the less radioactive hormone can bind to the antibody (Fig. 3A). The antibody-bound radioactivity is then separated from unbound radioactivity by a variety of physicochemical means and counted. The amount of hormone present in the plasma sample is inversely related to the amount of radioactivity recovered in the antibody complex and can be quantitated precisely by comparison with a standard curve constructed with known amounts of unlabeled hormone (Fig. 3B).

Sandwich-Type Assays

Once the amino acid sequence of a hormone is known, small peptides corresponding to specific regions of the hormone can be synthesized and used for producing antibodies that bind to specific portions of the hormone. If the hormone is large enough so that two different antibodies can bind to it without interfering with each other, we can obtain a "sandwich" in which the hormone becomes the "filling" linking two antibodies (Fig. 4). One antibody can be linked to a solid support such as a plastic dish with multiple wells. The plasma sample containing the hormone to be measured is then added and allowed to bind to the immobilized antibodies. A second "reporter" antibody linked to an enzyme such as a peroxidase or a phosphatase is then added, allowed to bind, and the excess washed away. A reaction is then run to quantitate the amount of reporter antibody retained by measuring a colored product. This scheme is one variation of the enzyme-linked immunosorbent assay (ELISA). Many other variations of this approach are also in use, some relying on a fluorescent moiety rather than an enzyme attached to the reporter antibody. Quantitation of hormone in the biologic sample is obtained by comparison with a standard curve constructed with known amounts of the hormone.

The major limitation of immunoassays is that immunologic rather than biologic activity is measured, because the portions of the hormone molecule recognized by the antibodies are unlikely to be the same as the portion recognized by the hormone receptor. Thus, a protein hormone that may be biologically inactive may

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