Radioimmunoassay

tions and adaptations of the original RIA, relying to a large degree on the principle of competitive binding on which the RIA is based. It is beyond the scope of this text to describe in detail the competitive binding assays currently used to measure hormone concentrations, but the principles are the same as those for the RIA.

The two key components of a RIA are a specific antibody (Ab) that has been raised against the hormone in question and a radioactively labeled hormone (H*). If the hormone being measured is a peptide or protein, the molecule is commonly labeled with a radioactive iodine atom (125I or 131I) that can be readily attached to tyrosine residues of the peptide chain. For substances lacking tyro-sine residues, such as steroids, labeling may be accomplished by incorporating radioactive carbon (14C) or hydrogen (3H). In either case, the use of the radioactive hormone permits detection and quantification of very small amounts of the substance.

The RIA is performed in vitro using a series of test tubes. Fixed amounts of Ab and of H* are added to all tubes (Fig. 31.6A). Samples (plasma, urine, cerebrospinal fluid, etc.) to be measured are added to individual tubes. Varying known concentrations of unlabeled hormone (the standards) are added to a series of identical tubes. The principle of the RIA, as indicated in Figure 31.6B, is that labeled and unlabeled hormone compete for a limited number of antibody binding sites. The amount of each hormone that is bound to antibody is a proportion of that present in solution. In a sample containing a high concentration of hormone, less radioactive hormone will be able to bind to the antibody, and less antibody will be able to bind to the radioactive hormone. In each case, the amount of radioactivity present as antibody-bound H* is determined. The response produced by the standards is used to generate a standard curve (Fig. 31.7). Responses produced by the unknown samples are then compared to the standard curve to determine the amount of hormone present in the unknowns (see dashed lines in Fig. 31.7).

^FGfflRSSnV^ The principles of radioimmunoassay (RIA).

^tammU^mmm a, Specific antibodies (Ab) bind with radioactive hormone (H*) to form hormone-antibody complexes (Ab-H*). B, When unlabeled hormone (open circles) is also introduced into the system, less radioactive hormone binds to the antibody. (Modified from Hedge GA, Colby HD, Goodman RL. Clinical Endocrine Physiology. Philadelphia: WB Saunders, 1987.)

One major limitation of RIAs is that they measure im-munoreactivity, rather than biological activity. The presence of an immunologically related but different hormone or of heterogeneous forms of the same hormone can complicate the interpretation of the results. For example, POMC, the precursor of ACTH, is often present in high concentrations in the plasma of patients with bronchogenic carcinoma. Antibodies for ACTH may cross-react with POMC. The results of a RIA for ACTH in which such an antibody is used may suggest high concentrations of ACTH, when actually POMC is being detected. Because POMC has less than 5% of the biological potency of ACTH, there may be little clinical evidence of significantly elevated ACTH. If appropriate measures are taken, however, such possible pitfalls can be overcome in most cases, and reliable results from the RIA can be obtained.

One important modification of the RIA is the radiore-ceptor assay, which uses specific hormone receptors rather than antibodies as the hormone-binding reagent. In theory, this method measures biologically active hormone, since receptor binding rather than antibody recognition is assessed. However, the need to purify hormone receptors and the somewhat more complex nature of this assay limit its usefulness for routine clinical measurements. It is more likely to be used in a research setting.

ELISA. The enzyme-linked immunosorbent assay

(ELISA) is a solid-phase, enzyme-based assay whose use and application have increased considerably over the past two decades. A typical ELISA is a colorimetric or fluoro-metric assay, and therefore, the ELISA, unlike the RIA, does not produce radioactive waste, which is an advantage, considering environmental concerns and the rapidly increasing cost of radioactive waste disposal. In addition, because it is

Radioimmunoassay Principle

^FGfflRSSnV^ The principles of radioimmunoassay (RIA).

^tammU^mmm a, Specific antibodies (Ab) bind with radioactive hormone (H*) to form hormone-antibody complexes (Ab-H*). B, When unlabeled hormone (open circles) is also introduced into the system, less radioactive hormone binds to the antibody. (Modified from Hedge GA, Colby HD, Goodman RL. Clinical Endocrine Physiology. Philadelphia: WB Saunders, 1987.)

Radioimmunoassay

kThe basic components of an ELISA. A typi-"cal ELISA is performed in a 3 X 5-inch plastic plate containing 96 small wells. Each well is precoated with an antibody (Ab1) that is specific for the hormone (H) being measured. Unknown samples or standards are introduced into the wells, followed by a second hormone-specific antibody (Ab2). A third antibody (Ab3), which recognizes Ab2, is then added. Ab3 is coupled to an enzyme that will convert an appropriate substrate (S) into a colored or fluorescent product (P). The amount of product formed can be determined using optical methods. After the addition of each antibody or sample to the wells, the plates are incubated for an appropriate period of time to allow antibodies and hormones to bind. Any unbound material is washed out of the well before the addition of the next reagent. The amount of colored product formed is directly proportional to the amount of hormone present in the standard or unknown sample. Concentrations are determined using a standard curve. For simplicity, only one Ab1 molecule is shown in the bottom of the well, when, in fact, there is an excess of Ab1 relative to the amount of hormone to be measured.

a solid-phase assay, the ELISA can be automated to a large degree, which reduces costs. Figure 31.8 shows a relatively simple version of an ELISA. More complex assays using similar principles have been developed to overcome a variety of technical problems, but the basic principle remains the same. In recent years the RIA has been the primary assay used clinically,- its use has expanded considerably, and it will likely be the predominant assay in the future because of the advantages listed above.

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