Other Assay Techniques

PPB. Parts per billion. One part per billion is a frequently used dimension for expressing the composition and analysis of substances—as found in air, water, food substances, etc. Instrument developments and other assay techniques perfected during the past decade or so have made the determination of such minute quantities a practical possibility for many materials. One part per billion is approximately equivalent to 1 drop in a 10,000-gall on (37,850-liter) tank. 

The principle of time-resolved fluorometry has been described in detail elsewhere in this book (refer to the chapter by Biinzli [50]) this chapter oidy concerns clinical applications of time-resolved fluorometric assay (TRFIA) and comparison of this method with other assay techniques. The sensitivity of TRFIA is based on the use of chelates of rare earth metals (lanthanides), e.g., emopium (Eu ), terbium (Tb" ), samarium (Sm" ), and dysprosium (Dy ). Of these, Eu usually provides the highest sensitivity and has therefore been most widely used [6]. The emissimi spectra and decay times of the lanthanides differ from each other, and it has therefore been possible to develop multilabel assays for simultaneous detectiOTi of up to four different antigens [7]. Simultaneous determination of several antigens is especially... 

ECL is often combined with other assay techniques to detect the concentration of a coreactant or an emitter more precisely. For example, in order to determine the precise coreactant concentration, ECL assays are used with the combination of some analytical methods for the separation of molecules such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) [9,10]. On the other hand, the detection of the ECL label as the emitter is often combined with magnetic bead-based assays (Fig. 3) that enables to... 

Once samples have been obtained, one can use a variety of available chemical, optical, spectroscopic, chromatographic, or other assays to determine concentration. For example, data in Figure 15-7 were obtained using a calibrated densito-metric technique in which one of the two species was colored in advance. Similar results have been obtained using other assay techniques, such as reflection near infrared spectroscopy to evaluate concentrations of magnesium stearate (a common pharmaceutical lubricant) or conductivity assays to evaluate the mixing of salt (NaCl, KCl) in anionic excipients (Avicel). 

The fluorescent labelling of heparin with F-D by this technique did not observably alter the biologic activity of the heparin as regards to its binding to antithrombin and catalysis of antithrombin s neutralization of activated coagulation factors. F-D labelled heparins also bound to other known heparin-binding proteins in a saturable and reversible manner, as demonstrated by the dot-blot assay technique (Figure 6). 

Additionally it has been our experience that mass spectrometry as a routine detection/identification technique for bacteria is not well received by microbiologists and clinicians who prefer less expensive, less complicated approaches to bacterial typing and identification, such as methods based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). For that reason we have adapted our MS approach to serve as a means of biomarker discovery that feeds candidate proteins or leads into development as PCR targets or other immunoassay techniques. 

The second label also may be a fluorescent compound, but doesn t necessarily have to be. As long as the second label can absorb the emission of the first label and modulate its signal, binding events can be observed. Thus, the two labeled DNA probes interact with each other to produce fluorescence modulation only after both have bound target DNA and are in enough proximity to initiate energy transfer. Common labels utilized in such assay techniques include the chemiluminescent probe, N-(4-aminobutyl)-N-ethylisoluminol, and reactive fluorescent derivatives of fluorescein, rhodamine, and the cyanine dyes (Chapter 9). For a review of these techniques, see Morrison (1992). 

Toxicity. There is only one Member State (Germany) having a test which is used to assess toxic hazards of combustion gases. The test is used mainly to evaluate non-combustible materials and is based on bio-assay techniques. The philosophies of other countries consider non-combustible materials as presenting no, or negligible toxic hazard. 

The establishment of quantitative methods for the determination of vitamins in body fluids and tissues by microbiological assay techniques should stimulate the search for the significance of vitamins in disease, not only in nutritional deficiency, but in the much wider field of all metabolic disturbances. Functional vitamin deficiencies are produced by malabsorption, by inhibitors of the vitamin function through products of the body, and particularly through drugs and other toxic substances. Vitamin deficiencies may be relative deficiencies whenever an individual s metabolism is deranged so as to require enhanced quantities of a given vitamin to cure or to counteract certain symptoms as, e.g., in Darier s disease (keratosis follicularis) (P2a). 

Cyclodiene pesticides, of which endrin and its oxidized analogs are representative, can also be estimated by receptor-assay technique. Cyclodiene pesticides exert their mode of action by altering central nervous system membrane ion transport. In work reported by Saleh et al. (1993), a labeled amino acid, GABA, that binds to the chloride channel receptor is displaced by endrin (and other similar molecules), and thus serves as an assay for these pesticides. The GABA receptor was shown to be a potentially useful biomarker for organochlorine pesticides such as lindane, toxaphene, endrin, chlordane, and others. The assay involves small quantities of blood (0.1 mL), and requires only that the plasma be separated from the... 

In stability testing, HPLC is the most frequently used chromatographic technique for assay. HPLC has illustrated the benefits of speed, resolution, accuracy, and sensitivity, and HPLC can interface with many detectors that other analytical techniques are unable to offer. 

The classical microbial assay approaches to measuring antibiotic residues, diffusion, turbidimetric and acid production were described and the advantages and limitations reviewed. Other systems so discussed and reviewed were the affinity or receptor methods and the immunological approach using ELISA or EMIT assay techniques. The classical systems, in general, could measure antibiotic residues at the fractional ppm to the ppb levels. The potentials of the receptor and immunological assay system were discussed. 

Two example-determinations of TTX using fluorometry coupled with high-performance liquid chromatography (HPLC) are introduced here, along with some other promising assay techniques such as mass spectrometry. 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Water in food products can be described as being free or bound. The definition of what consitiutes bound water is far from clear (see Fennema, 1985) but it can be considered as that part of the water in a food which does not freeze at — 40°C and exists in the vicinity of solutes and other non-aqueous constituents, has reduced molecular mobility and other significantly altered properties compared with the bulk water of the same system (Fennema, 1985). The actual amount of bound water varies in different products and the amount measured is often a function of the assay technique. Bound water is not permanently immobilized since interchange of bound water molecules occurs frequently. 

Biochemical research often requires the quantitative measurement of protein concentrations in solutions. Several techniques have been developed however, most have limitations because either they are not sensitive enough or they are based on reactions with specific amino acids in the protein. Since the amino acid content varies from protein to protein, no single assay will be suitable for all proteins. In this section we discuss five assays three older, classical methods that are occasionally used today and two newer methods that are widely used. In four of the methods, chemical reagents are added to protein solutions to develop a color whose intensity is measured in a spectrophotometer. A standard protein of known concentration is also treated with the same reagents and a calibration curve is constructed. The other assay relies on a direct spectrophotometric measurement. None of the methods is perfect because each is dependent on the amino acid content of the protein. However, each will provide a satisfactory result if the proper experimental conditions are used and/or a suitable standard protein is chosen. Other important factors in method selection include the sensitivity and accuracy desired, the presence of interfering substances, and the time available for the assay. The various methods are compared in Table 2.3. 

Direct determination of urea pesticides by high-performance liquid chromatography has been widely reported in the literature (10,32-36,127-130). Ultraviolet detection has often been used (32,33,35,36,60,127) with usually acceptable sensitivity, although this detector is nonspecific and the sensibility is, in general, low. To overcome this problem, several techniques have been assayed, such as precolumn enrichment (60), postcolumn derivatization (34,10), and the use of other detection techniques such as the electrochemical (129), photoconductivity (128,130), and fluorescence detectors (9,10,34). Table 9 summarizes representative papers using these techniques in HPLC analysis. 

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