Assay formats unlabelled

Most modem RJAs utilize a competitive assay format (Fig. 2) in which radiolabled antigen, Ag, competes with unlabeled antigen, Ag, in a sample for binding to the antibody. Ah. The free antigens are then separated from the antigen—antibody complexes, and the amount of radioactivity in the... 

Immunoassays can be classified according to different criteria. The particular type selected has a strong influence on the assay performance with regard to precision and sensitivity. The main criteria include [3, 22, 23] (1) labeled or unlabeled assay formats, with different type of labels (2) competitive or non-competitive immunoassays, and (3) homogeneous or heterogeneous immunoassays. These classifications can also be extended to MIP-ILAs (Fig. 2) ... 

In principle, this miniaturized competitive assay format for unlabelled enzyme inhibitors or substrates could be used to profile many different drug-like molecules in parallel, or alternatively to determine the binding constants of an individual drug-like molecule by making kinetic measurements at varying concentrations of the test compound. 

The lipopolysaccharide (LPS) endotoxin is the most powerful immune stimulant known and a causative agent in the clinical syndrome known as sepsis. Sepsis is responsible for more than 100,000 deaths annually, in large part due to the lack of a rapid, reliable, and sensitive diagnostic technique. An evanescent wave fiber-optic biosensor was developed for the detection of LPS from E. coli at concentrations as low as lOngmL in 30 s (James et al., 1996). Polymyxin B covalently immobilized onto the surface of the fiber-optic probe was able to bind fluorescently labeled LPS selectively. Unlabeled LPS present in the biological samples was detected in a competitive assay format, by displacing the labeled LPS. The competitive assay format worked in buffer and in plasma with similar sensitivities. This method might also be used with other LPS capture molecules, such as antibodies, lectins, or antibiotics, to simultaneously detect LPS and determine the LPS serotype. 

The competitive assay is another format used to quantitate an analyte. An unlabeled analyte competes with a labeled analyte (enzyme-conjugated molecule) for binding to a specific capture antibody (Figure 11.1c). 

Several successful attempts were done to transfer classical CEIA to a microchip-based format. This kind of miniaturization is a trend that can overcome the limitations of CE in high-throughput systems. On-chip CE offers both parallel analysis of samples and short separation times. Koutny et al. showed the use of an immunoassay on-chip (32). In this competitive approach fluorescein-labeled cortisol was used to detect unlabeled cortisol spiked to serum (Fig. 8). The system showed good reproducibility and robustness even in this problematic kind of sample matrix. Using serum cortisol standards calibration and quantification is possible in a working range of clinical interest. This example demonstrated that microchip electrophoretic systems are analytical devices suitable for immunological assays that can compete with common techniques. 

The attachment of protein to the transducer surface for immunoassay is a difficult problem, since it must be achieved without interfering with the active site. If an unlabeled assay is to be performed it must also be such that subsequent non specific interaction with the surface can be inhibited. This requirement is often contrary to those for effective antibody-antigen complex formation. A surface close packed with antibody will be sterically hindered and its reaction with antigen inhibited. On the other hand, a suitably spaced packing allows non-specific interactions to occur and large false positive signals to be recorded. As mentioned earlier, this was demonstrated by Cullen and Lowe who used the surface plasmon resonance technique to probe specific and non-specific protein interactions at metal surfaces. (23). 

Fig. 5 NRL Array Biosensor mixed format immunoassays (modified from [119]). Schematic of (a) the sandwich and (b) the competitive immunoassay fonnats used in the detection of the Campylobacter jejuni and aflatoxin Bi (AFBi), respectively, (a) Sandwich format antigen captured by the immobilized antibody then quantified by passing a second, fluorescently labeled, antibody over the surface, (b) Competitive format competition for binding sites on the fluorescently labeled antibody occurs between the unlabeled antigen in solution and the surface-bound antigen analog, (c) Final charge-coupled devices image taken with the NRL Array Biosensor of a waveguide exposed simultaneously to the C. jejuni (5 x 10" cfu/mL) sandwich assay (SAND) and the aflatoxin Bj (AFBi-1 ng/mL) competitive assay (COMP) in various combinations...

The mechanism of osazone formation has been studied by the isotopic-tracer technique, by using an N -labeled arylhydrazone, treating it with an unlabeled arylhydrazone, and making an isotopic assay of the reaction products. The results were characteristic of Weygand s mechanism involving the oxidation of the hydrazone to a l-imino-lV -2-keto derivative (40) and subsequent osazone formation with the elimination of ammonia-iV . 

Having established the means to monitor the catalytic turnover of a fluorogenic substrate by a CYP450 enzyme in a microarray format, it is then relatively straightforward to set up competitive assays to determine the kinetic parameters for either unlabelled inhibitors or unlabelled substrates as follows (see Note 18) ... 

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