Immunoassay clinical development

While the clinical use of MS/MS of hormonal steroids is new, metabolite analysis by gas chromatography (GC)-mass spectrometry (MS) has been available for 40 years, since few immunoassays were developed for urinary analytes. Profile analysis is a very powerful technique and it must be recognized that with few exceptions, all disorders of steroid synthesis and metabolism first had their metabolome defined... 

As for any bioanalytical method, the extent of validation for an immunoassay should be related to the intended application of the assay. Thus, if an immunoassay is intended to support rapid screening in discovery R D, the characterization of specificity and the accuracy and precision specifications may be less stringent than if the assay is used to support pre-clinical and clinical development studies. Indeed, an assay for discovery support may be designed to detect active metabolites as well as parent molecule, so that... 

Six CMIA mono-immunoassays were developed (cf Table 8.1). The first of these were in the area of clinical biology, with CMIA assays of antiepileptic medications such as carbamazepine [45], phenobarbital [71], diphenylhydantoin [26, 32], and a steroid hormone, cortisol [70]. More recently they have been extended into the environmental area, with assays of pesticides atrazine [44] and chlortoluron [39]. All these assays used polyclonal antibodies. For each of the analytes dilution curves and standard curves were obtained. The curves obtained for carbamazepine, representative of the results for all compounds, are shown in Fig. 8.8. 

Antibodies have been generated which produce immunoassays that discriminate between GH-V and GH-N. These assay systems have shown that the secretion of GH-V becomes elevated at about three weeks of pregnancy and increases to approximately 15 ng/mL near term (8). The physiological role of GH-V is uncertain. Genetic deficiency of GH-V does not adversely affect pregnancy or fetal development (9). GH-V is a potent growth-stimulator but possesses considerably less lactogenic activity than GH-N (10). There are no clinical appHcations (ca 1993) for GH-V. 

Immunodiffusion and immunoprecipitation, developed ia the 1940s as a means to identify and semiquantitate specific proteias, were the direct precursors to the development ia 1953 of Immunoelectrophoresis, a method used ia many clinical laboratories (5). Single- and double-gel immunodiffusion and immunoelectrophoresis were, ia effect, the first standardized and routinely used immunoassay methods (see Electroseparations, electrophoresis). 

EIAs can be used per se or with a spectrophotometer. Traditionally, EIAs have been developed in 96-weU microtiter plates which provide the immobilization support for the assay, the reaction vessel, and, when linked to a spectrophotometer-based reader, a rapid means to detect and quantify the color resulting from interaction of a substrate with the antibody—antigen—enzyme complex. Automated immunoassay analyzers targeted primarily for use in the clinical laboratory have taken automation one step further, utilizing robotics to carry out all reagent additions, washings, and final quantification including report preparation. 

The singularity of MAbs and the ease of mass production appeared to be the answer to rapid development of highly specific immunoassays. Companies were formed to produce MAbs and incorporate them into assays. In fact, such assays have been developed and have proved very successful for infectious diseases, hormones, and other clinical analytes. 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Two immunoassays have been developed to measure tryptase in human fluids, one that measures mature a/(3-tryptases, i.e. total tryptase, available commercially, and one developed by Schwartz et al. [7] that measures both mature (3-tryptase and immature a/(3-tryptases. This distinction is of clinical relevance since immature tryptases reflect mast cell burden whereas mature tryptases indicate mast cell activation. Thus, for the diagnosis of anaphylaxis it would be extremely important to be able to differentiate between acute anaphylaxis and increases in tryptase due to increase in numbers of mast cells as happens in mastocytosis. Total tryptase would be high in both conditions, whereas mature tryptase will be only high in anaphylaxis but negligible in mastocytosis. 

The development of sensitive and inexpensive immunoassays for low molecular weight pesticides has been an important trend in environmental and analytical sciences during the past two decades. 0.27-29 jq design an immunoassay for a pesticide, one can rely on the immunoassay literature for agrochemicals, " but many of the innovations in clinical immunoanalysis are also directly applicable to environmental analysis. - Conversely, the exquisite sensitivity required and difficult matrices present for many environmental immunoassay applications have forced the development of technologies that are also useful in clinical immunoassay applications. In the following discussion we will describe widely accepted procedures for the development of pesticide immunoassays. 

The analysis of fish tissues for ciguatoxin by a newly developed enzyme-immunoassay procedure (26, 27) has been carried out in this study. Three areas of examinations have been attempted (1) the examination of clinically defined and documented and non-toxic consumed fish samples (2) the assessment of freshly caught fishes from the sites in the Leeward part of the island of Oahu where ciguatoxin is found and (3) competitive inhibition with suspension of purified ciguatoxin and closely related structurally similar polyether toxins. 

Two recent developments of immunoassay, namely the use of free radicals and enzymes (M17, RIO) as markers instead of isotopic nuclides, can be expected to increase the scope and application of the technique. Both have already been applied to the detection and measurement of drugs in biological fluids but have not, so far, achieved the requisite specificity and precision necessary for clinical use. 

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