Radioimmunoassays,

Fignre 8-32. Conceptual operation of the radioimmunoassay, RIA. The soluble Ab-Ag complex is both a product of the first step and a reactant in the second step of the assay. 

Hormone lodination Procedure I Atoms/Hormone Molecule Methods of Separation of lodohormone from Uniodinated Hormone Reference 

Adrenocorticotropin Tracer iodination with chloramine T 0.01 Chromatography on carboxymethyl-cellulose column salt gradient, ammonium acetate R. J. Lefkowitz, J. Roth, W. Pricer, and I. Pastan, Proc. Natl. Acad. Sci. US, 65 745 (1970) 

Insulin Tracer iodination with chloramine T 0.025-0.10 Chromatography on diethylamino-ethyl cellulose column salt gradient, Tris-NaCl-urea P. Freychet, J. Roth, and D. M. Neville, Jr., Biochem. Biophys. Res. Commun., 43 400 (1971) 

Luteinizing hormone, releasing hormone Lactoperoxidase 0.02 Polyacrylamide gel electrophoresis Y. Miyachi, A. Chrambach, R. Mecklenburg, and M. B. Lipsett, Endocrinology, 92 1725 (1973) 

Despite the high sensitivity and precision of RIA, the method has largely been replaced by ELISA because of concerns about the use of radioactive isotopes. 

The mixed anhydride method was used with diaminoethane to give a free amino group that was subsequently reacted with 4-hydroxybenzoic acid to provide an aromatic ring to allow iodination with [ ] to increase sensitivity of the assay compared with labelled radioactive tracers. This reaction could also be used to prepare fluorescent compounds for cell-biology studies. 

ASBT apical sodium-dependent bile-acid transporter 

IBABP intestinal bile-acid-binding protein 

SREBP sterol regulatory element binding protein 

Proteins from animal species are usually designated with the first letter upper case and subsequent letters in lower case, while those from man are all in upper case. For simplicity all upper case is used generically unless an animal source is described. 

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Human intestinal mucosa as weU as livei can peifomi this conversion (344,345). Desogestiel and 3-ketodesogestiel can be measured by radioimmunoassay or hplc (339,345). 

Medroxyprog esteroneAcetate. Accurate pharmacokinetic and metaboHsm studies on MPA have been difficult because the radioimmunoassays employed caimot differentiate between MPA and its metaboHtes (346). Comparison of MPA plasma levels assayed by hplc and radioimmunoassay show that radioimmunoassay may overestimate intact MPA concentrations by about fivefold (347). However, values of the mean elimination half-life of MPA were similar, being 33.8 and 39.7 h when measured by hplc and radioimmunoassay, respectively (347). Approximately 94% of MPA in the blood is bound to albumin (348). When taken orally, MPA is rapidly absorbed with Htde or no first-pass metaboHsm (13). Peak semm levels ate reached after 3 h. Steady state occurs after three days of daily adininistration (349). The pharmacokinetics of MPA when adininistered in a depot formulation have been described (350). 

Mifepristone. After oral adininistration, peak plasma levels of mifepristone (84) (RU 486) are reached in 1 h and over 95% was bound to plasma proteins (351,352). The plasma half-life of RU 486 is approximately 24 h (352,353). In humans, monodemethylated (98), didemethylated (99) and alcohoHc nondemethylated (100) metaboHtes of RU 486 have been identified (351). These metaboHtes show some progestin-binding affinity, approximately five to ten times lower than that of RU 486 itself. RU 486 and its metaboHtes can be measured by radioimmunoassay and hplc (353,354). 

Radiotracers have also been used extensively for the quantitative rnicrodeterrnination of blood semm levels of hormones (qv), proteins, neurotransmitters, and other physiologically important compounds. Radioimmunoassay, which involves the competition of a known quantity of radiolabeled tracer, usually I or H, with the unknown quantity of semm component for binding to a specific antibody that has been raised against the component to be deterrnined, is used in the rnicro deterrnination of physiologically active materials in biological samples (see Immunoassay). 

A definitive method for stmctural deterrnination is x-ray crystallography. Extensive x-ray crystal stmcture deterrninations have been done on a wide variety of steroids and these have been collected and Hsted (270). In addition, other analytical methods for steroid quantification or stmcture determination include, mass spectrometry (271), polarography, fluorimetry, radioimmunoassay (264), and various chromatographic techniques (272). 

Body fluids are analyzed for T and T by a variety of radioimmunoassay procedures (31) (see Immunoassays). The important clinical parameter for estimating thyroid function, the protein-bound iodine (PBI), is measured as described in treatises of clinical chemistry. High performance Hquid chromatographic (hplc) methods have replaced dc (32,33). 

The immunochemical interaction between the antigen and antibody is very specific. By labeling either the antigen or antibody, the method s sensitivity is increased. The most frequently used labels to increase sensitivity are radionucHdes (see Radioisotopes) where the assay process is called radioimmunoassay (RIA), or en2ymes where the assay is named en2yme immunoassay (ElA) (see Enzyme applications). 

Specific IgE Assay. Two radioimmunoassays are available in France using a quaternary ammonium compoimd coupled to Sepharose [30, 31]. The sensitivity of these tests was equivalent at 88%, the specificity reaches 90%. A morphine-based immunoassay has been proposed in Australia [14]. More recently, Ebo et al. [32] investigated a rocuronium ImmimoCAP and set the sensitivity at 85%, the specificity being absolute, provided an assay-specific decision threshold is applied. An ImmimoCAP (Phadia A) is available. 

Mertes PM, Moneret-Vautrin DA Skin reactions to intradermal neuromuscular blocking agent injections a randomized multicenter trial in healthy volunteers. Anesthesiology 2007 107 245. Moneret-Vautrin DA, Gueant JL, Kamel L, Laxenaire MC, el Kholty S, Nicolas JP Anaphylaxis to muscle relaxants cross-sensitivity studied by radioimmunoassays compared to intradermal tests in 34 cases. J Allergy Clin Immunol 1988 82 745. 

Guilloux L, Ricard-Blum S, Ville G, Motin J A new radioimmunoassay using a commercially available sohd support for the detection of IgE antibodies against muscle relaxants. J Allergy Clin Immunol 1992 90 153. 

Ferritin is another protein that is important in the metabolism of iron. Under normal conditions, it stores iron that can be called upon for use as conditions require. In conditions of excess iron (eg, hemochromatosis), body stores of iron are greatly increased and much more ferritin is present in the tissues, such as the liver and spleen. Ferritin contains approximately 23% iron, and apoferritin (the protein moiety free of iron) has a molecular mass of approximately 440 kDa. Ferritin is composed of 24 subunits of 18.5 kDa, which surround in a micellar form some 3000-4500 ferric atoms. Normally, there is a little ferritin in human plasma. However, in patients with excess iron, the amount of ferritin in plasma is markedly elevated. The amount of ferritin in plasma can be conveniently measured by a sensitive and specific radioimmunoassay and serves as an index of body iron stores. 

Human erythropoietin is a glycoprotein of 166 amino acids (molecular mass about 34 kDa). Its amount in plasma can be measured by radioimmunoassay. It is the major regulator of human erythropoiesis. Erythropoietin is synthesized mainly by the kidney and is released in response to hypoxia into the bloodstream, in which it travels to the bone marrow. There it interacts with progenitors of red blood cells via a specific receptor. The receptor is a transmembrane protein consisting of two different subunits and a number of domains. It is not a tyrosine kinase, but it stimulates the activities of specific... 

Methods of detection, metabolism, and pathophysiology of the brevetoxins, PbTx-2 and PbTx-3, are summarized. Infrared spectroscopy and innovative chromatographic techniques were examined as methods for detection and structural analysis. Toxicokinetic and metabolic studies for in vivo and in vitro systems demonstrated hepatic metabolism and biliary excretion. An in vivo model of brevetoxin intoxication was developed in conscious tethered rats. Intravenous administration of toxin resulted in a precipitous decrease in body temperature and respiratory rate, as well as signs suggesting central nervous system involvement. A polyclonal antiserum against the brevetoxin polyether backbone was prepared a radioimmunoassay was developed with a sub-nanogram detection limit. This antiserum, when administered prophylactically, protected rats against the toxic effects of brevetoxin. 

Standard curves performed under our defined radioimmunoassay conditions ([ H]PbTx-3 = 1 nM, antiserum dilution = 1 2000, assay volume = 1 ml) demonstrated the ability of this antiserum to bind equally to PbTx-2 and PbTx-3, suggesting specificity for the cyclic polyether backbone region of the molecule (Figure 8). The linear portion of the curve indicated a lower detection limit of 0.2-0.5 ng in saline buffer under these conditions. Evaluation of this assay for use with biological fluids and tissue extracts is underway. 

Figure 8. Standard curves for PbTx-2 ( ) and PbTx-3 ( ) in the brevetoxin radioimmunoassay. Lower detection limits are 0.2 — 0.5 ng in phosphate-buffered saline (PBS). Standard RIA conditions [ H]PbTx-3 = 1 nM antiserum dilution = 1 2000 sample vol. = 1 ml buffer = 0.1 M PBS, pH = 7.4.

Cultures of Gambierdiscus toxicus have been obtained in several laboratories (14,17-19). These cultures produce large amounts of maitotoxin and low amounts of lipid-soluble CTx-like toxin. However, in most cases, this toxin has not been unequivocally identified as CTx. The only firm evidence that cultures of Gambierdiscus toxicus produce CTx was provided by Baden et al. (20) who used radioimmunoassays and electrophysiological experiments to characterize the toxin. It is possible that cultured Gambierdiscus toxicus produce only trace amounts of CTx and that levels of production comparable to those found in natural populations are dependent on yet undefined environmental parameters. 

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