Hormone antibody specificity

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

Fermentation broths are complex, aqueous mixtures of cells, comprising soluble extracellular, intracellular products and any unconverted substrate or unconvertible components. Recovery and extraction of product is important in bioprocess engineering. In particular separation is a useful technique it depends on product, its solubility, size of the process, and product value. Purification of high-value pharmaceutical products using chromatography such as hormones, antibody and enzymes is expensive and difficult to scale up.1 Tire necessary steps to follow a specific process depend on the nature of the product and the characteristics of the fermentation broth. There are a few steps for product recovery the following processes are discussed, which are considered as an alternative for product recovery from fermentation broth. 

A second unanswered concern is whether the antibody induced by the recombinant protein has any discernible health effect. Other than some reports of neutralization of biological activity, little pathology has been attributed to the presence of antibodies in patients given recombinant protein therapy. It should also be noted that the question of antibody specificity has not been well studied, so that it is entirely conceivable that autoimmune pathology or even an anaphylaxis response could be induced. Equally important is the concern that induced antibody might neutralize the endogenous hormone or protein that it is intended to replace or supplement. 

It is well known that a very important feature of many biological systems is specific recognition at the molecular level. Antibodies as a group are widely used for molecular recognition, e.g., affinity assays. This feature can be used by labeling an anti-human growth hormone antibody fraction with a fluorescent tag... 

FIGURE 23-3 Radioimmunoassay (RIA). (a) A low concentration of radiolabeled hormone (red) is incubated with (T) a fixed amount of antibody specific for that hormone or (2) a fixed amount of antibody and various concentrations of unlabeled hormone (blue). In the latter case, unlabeled hormone competes with labeled hormone for binding to the antibody the amount of labeled hormone bound varies inversely with the concentration of unlabeled hormone present, (b) A radioimmunoassay for adrenocorticotropic hormone (ACTH). A standard curve of the ratio [bound] to [unbound radiolabeled ACTH] vs. [unlabeled ACTH added] is constructed and used to determine the amount of (unlabeled) ACTH in an unknown sample. If an aliquot containing an unknown quantity of unlabeled hormone gives, say, a value of 0.4 for the ratio [bound]/[unbound] (see arrow), the aliquot must contain about 20 pg of ACTH. 

A major application of monoclonal antibodies is in clinical assays for drugs, bacterial and viral products, tumor antigens, hormones, and other circulating proteins. Their use in conjunction with immunoassays (Box 31-C) has provided increased specificity and sensitivity. Another major application is to observe binding of antibodies to specific proteins by electron microscopy. The location of specific receptor proteins can be established - as can the locations of ribosomal proteins and many other cellular components (Fig. 29-1). Monoclonal antibodies to acetylcholine receptors have been shown to induce symptoms of myasthenia gravis (Box 31-D), supporting the autoimmune origin of that disease. 1 Monoclonal antibodies specific for such a small hapten as mercuric ion have been isolated.k... 

Wl. Wada, H. G., Danisch, R. J., Baxter, S. R., Federici, M. M., Fraser, R. C., Brownmill-er, L. J., and Lankford, J. C., Enzyme immunoassay of the glycoprotein tropic hormones—choriogonadotropin, lutropin, thyrotropin—with solid-phase monoclonal antibody for the a-subunit and enzyme-coupled monoclonal antibody specific for the fi-subunit. Clin. Chem. 28, 1862-1866 (1982). 

Antibodies specific for a hormone erythropoietin, are being tested as a monitor for hormone therapy of anemia. The antibody can be used to identify the misuse of the hormone by athletes to enhance performance in competitive sports. 

Haptens.—Haptens have been prepared from steroid hormones by reactions at C-3 or C-17, or in the pregnane side-chain. Antibody specificity is often improved, however, by anchoring the steroid through a middle-ring site to the protein, so that both ends of the steroid component of the complex are exposed for recognition in the antibody-forming process. 

In order to assay gonadotropins by immunological methods, it is essential not only to have purified samples of FSH and LH, but also to possess antisera specific for these hormones. The preparation of purified FSH and LH has already been discussed. Accordingly it now remains to examine the question of antibody specificity in relation to the assay of these hormones. 

This variation in antibody specificity with successive inoculations of an animal has proved a matter of considerable diflBculty in the field of the radioimmunoassay of the gonadotropic hormones. The work of Odell et al. (03) demonstrates that, during the production of their antisera to human FSH, their experience was similar to that of Benjamini et al. (B4), namely, that early in immunization the population of antibodies which reacted with both FSH and LH was 100 times... 

One of the major aims of this review has been to examine the current status of immunological assays used to measure the pituitary gonadotropic hormones. Since all immunoassays basically depend on the interaction of an antigen of unique identity (in this case FSH or LH) and its specific antibody, it was necessary also to discuss the chemical nature of the various preparations of FSH and LH which are currently available and to consider the concept of antibody specificity as it applies in this field. 

Although other workers have prepared and used antisera in immuno-fiuorescence studies, confirmatory reports of the practicability of preparing antisera suitable for radioimmunoassays have not yet appeared. A considerable degree of cross-reactivity between species, both in the effects of administered hormone and in hormone-antibody interactions, appears to be a feature of most thyrocalcitonin preparations so far studied. Although this partial lack of species specificity should facilitate the development of antisera and of isotopically labeled hormone preparations from animals for use in immunoassays in man, it must also leave some doubt with respect to the specificity of such assays when applied to so complex a mixture as blood plasma. 

A EXPERIMENTAL FIGURE 11-43 Fusion proteins from expression vectors demonstrate that the hormone-binding domain of the glucocorticoid receptor (GR) mediates translocation to the nucleus in the presence of hormone. Cultured animal cells were transfected with expression vectors encoding the proteins diagrammed at the bottom. Immunofluorescence with a labeled antibody specific for p-galactosidase was used to detect the expressed proteins in transfected cells, (a) In cells that expressed p-galactosidase alone, the enzyme... 

Fig. 43.12. Standard curve for a radioimmunoassay. A constant amount of radioactive T4 is added to a series of tubes, each of which contains a different amount of nonradioactive T4. The amount of radioactive hormone that binds to an antibody specific for the hormone is measured and plotted against the nonradioactive hormone concentration. When more nonradioactive T4 is present in the tube, less radioactive T4 binds to the antibody.

Specificity The property of an enzyme, hormone, antibody, receptor, etc. to be extremely selective in recognising and attaching to another substance. 

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