Hormonal analyses. Most hormone assays performed today are of the competitive-binding variety. For a competitive-binding assay to be of value it must be practical and reliable. Criterion of reliability are defined in Table 2-7.
The RIA is the conventional prototype of a competitive-binding assay. There are three fundamental components to the RIA - radioactive ("hot") hormone, unlabeled ("cold") hormone (standard or sample), and antibody. Radioisotopes of tritium (b emitter) and iodine (high specific activity g emitter) are incorporated into steroid and protein (Tyr or His residues) hormones, respectively; this must be done without significant damage to the immunoreactivity of the hormone. Tracer and standard or unknown sample compete for a limited number of binding sites on the antibody. Amounts of (excess) tracer and antibody for each reaction are held constant, while quantities of standard hormone are increased step-wise. Reactions are allowed to proceed to equilibrium, and free (unbound) hormone is segregated from antibody-hormone complexes. Emission of energy from the bound complex is monitored by radiation detection equipment. As content of standard is increased from 0 (ie., 100% of antibody is bound by tracer), the amount of antibody-bound tracer declines reciprocally - a standard curve is constructed from these data. Reaction tubes containing sample in place of standard are assayed simultaneously. Estimates of mass of hormone within a sample are interpolated from the standard curve (Figure 2-38).
Antibodies belong mainly to the gamma globulin (IgG) class of immunoglobulins. Each Fab arm of the (bivalent) antibody can bind a molecule of ligand. Binding is mediated by weak noncovalent forces (ionic interactions, hydrogen bonding, hydrophobic attractions, van der Waals attraction); therefore, like that of enzyme-substrate binding, the reaction is reversible.
Antisera can be generated by injecting purified hormone into a species of animal that is capable of mounting an immunological reaction to that hormone (ie., do not produce the hormone in a chemical form that is exactly similar). Some small molecules (haptens) are not antigenic on their own (eg., steroid and peptide hormones, prostaglandins) and must first be coupled (at a nonactive site) to an immunogenic carrier (eg., albumin, keyhole limpet hemocyanin) before injection.
Even under the best of conditions of immunization, antisera can contain antibodies (polyclonal) that cross-react with related substances - the development of technology using monoclonal (homogenous) antibodies has helped in this respect. To obtain monoclonal antibodies an animal (eg., mouse) is injected with purified antigen, spleen cells capable of secreting a single type of antibody (clones) are screened and isolated, and selected cells are fused with myeloma (immortal) cells to produce a hybridoma. Cells maintained in culture provide a continuous source of antibody. A single hybridoma can yield approximately 1000 specific molecules of antibody per second.
A convenient method to separate antibody-hormone complexes from free hormone is to adhere the antibody to a solid phase, such as to the walls of a test tube. The free hormone can then be decanted (a centrifugation step is not required). Because proteins attach nonspecifically to plastic (eg., polyvinyl chloride or polystyrene), tubes can be coated by simply incubating with a solution containing antibody. Remaining unoccupied sites are then filled with an irrelevant protein, such as serum albumin or gelatin. One criticism of antibody-coated tubes is adsorption can mask immunoreactive (Fab) sites: to overcome this problem, protein A, a molecule derived from staphylococcus aureus that binds the Fc tail of IgG, can be coated to the solid phase (this permits extraction of IgG from the fluid-phase reaction mixture). Alternatively, precipitation of hormone-antibody complexes can be achieved using ammonium sulfate, magnetically-activated antibody, or with a second antibody generated against the first antibody (ie., anti-IgG). Adsorption of free (low molecular weight ligand) can be achieved with dextran-coated charcoal.
Other analytical systems that exploit the same basic principle as the RIA include the protein-binding assay, radioreceptor assay (RRA), scintillation proximity assay (SPA), enzyme immunoassay (EIA), fluoroimmunoassay (FIA), and chemiluminescent assay (CIA). Protein-binding and radioreceptor assays are radioligand assays that utilize an endogenous plasma protein (eg., for steroid hormones) or cellular receptor, respectively - instead of an antibody. Protein-binding assays lack the specificity of an immunoassay. The radioreceptor assay has an advantage over the RIA in that it only detects bioactive hormone (ie., antibodies can interact with sites on the hormone molecule not involved in receptor binding). Notwithstanding, it is difficult to isolate abundant quantities of stable receptor for routine analyses. Fortunately, data obtained from RIAs and RRAs are usually comparable.
A newly-developed methodology, SPA, does not require separation of bound from free ligand. Competitive binding of labeled ligand in proximity to antibody- or receptor-coated fluoromicrospheres allows the energy emitted to excite the fluor and produce detectable light that can be measured in a scintillation counter without liquid cocktail. Unbound tracer is too far from the microsphere to enable energy transfer before it is absorbed by the aqueous solution.
In the EIA, FIA ,and CIA, radioactive hormone is replaced by an enzyme-, fluorescein- or luminol-tagged ligand, respectively. Quantification is accomplished with a fluorometer in FIA and a luminometer in CIA. In EIA an extra step is required first - addition of substrate. An example of an enzyme commonly used in enzyme immunoassays is horseradish peroxidase: hydrogen peroxide (substrate) is reduced by this enzyme, and in the process an appropriate hydrogen donor (eg., o-phenylenediamine) is oxidized, causing a change in color of solution - appearance of product is measured by spectrophometric analysis of color reactions (ie., absorbance) to graded concentrations of hormone.
Antibody-excess immunoassays include the immunoradiometric assay (IRMA) and enzyme-linked immunosorbent assay (ELISA). In the IRMA cold ligand is "sandwiched" between an antibody coated to a solid phase and a second radiolabeled antibody raised against a different hormonal epitope (this works best with macromolecular hormones); sensitivity is not mandated by competition, and therefore, reactions can be carried out expeditiously over a wide range of detection. In a sandwich ELISA, hormone is bound to an antibody attached to a solid phase, and then an antibody-enzyme conjugate and substrate are added (Figure 2-39). These methods engender a direct relationship between radioactivity measured in the final complex and concentration of standard or analyte (in contrast to the inverse correlation between bound radioactivity and standard or sample concentrations in an RIA).
Nonradioisotopic procedures, such as ELISAs, are becoming popular because of lowered equipment costs, reduced hazard to users and the environment (ie., associated with handling and disposal of radionuclides), and can be adapted (subjective appraisal of color-change) for in-the-home or on-the-farm/ranch diagnostics. However, ELISAs tend to be less sensitive than the RIA (Table 2-8).
A reverse hemolytic plaque assay is used to detect secretion of hormone from individual cells (eg., gonadotropes) contained within a heterogeneous population. The concept is that a secretory product of a cell can be measured by specific antibodies in the presence of erythrocytes coated with protein A and added complement. Interaction of hormone with binding sites on the antibody causes stearic alterations in the antibody allowing for fixation of complement by juxtaposed Fc. Complement-induced hemolysis leads to the formation of a clear zone of erythrocyte membrane "ghosts" (ie., a plaque) surrounding the secretory cell (Figure 2-40). The plaque technique is sensitive and areas of lysis can be quantitated.
Receptor analyses. It is technically more difficult to monitor changes in populations of hormonal receptors than to evaluate alterations in patterns of secretion of hormones; yet, knowledge of dynamics of cellular receptors is no less important (eg., in diseases of endocrine resistance). The task of receptor measurement can be accomplished by exposing a constant amount of receptor (eg., tissue homogenate) to increasing concentrations of radioactive hormone. Receptor bound with hormone is separated from free radiolabel and each fraction is counted - the Scatchard plot is a common method of data assessment (Figure 2-41). Receptors not occupied by hormones are generally characterized unless special methods are first used to elute endogenous ligand from its binding site.