Probe assay-chemiluminescence enhanced

Ch. 31. PACE (Probe Assay—Chemiluminescence Enhanced) A Magnetic Bead Assay for Noncultural Diagnosis of Gonorrhea,... 

Chemiluminescence assays are ultrasensitive (attomole to zeptomole detection limits) and have wide dynamic ranges. They are now widely used in automated immunoassay and DNA probe assay systems, (e.g., acridinium ester and acri-dinium sulfonamide labels and 1,2-dioxetane substrates for alkaline phosphatase labels and the enhanced-luminol reaction for horseradish peroxidase labels [see Chapter 9]). 

Use of nonradioactive substitutes such as scintillation proximity assays for P or sulfur-35 ( S) sequencing studies or cation assays, and enhanced chemiluminescence (ECL) as a substitute for P and DNA probe labeling and southern blot analysis. 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

In work similar to that described in Section 20.2.5.10., Haupt and co-workers have found yet another use for their 2,4-D-imprinted polymer [51]. Whereas in the work described above the competing analyte was CMMC (see Fig. 20.14), in these experiments 2,4-dichlorophenoI (DCP) was used as the competitor. This compound is an enhancer of the peroxidase-catalysed chemiluminescence of the well known reaction of luminol and H2O2. Thus, in a certain concentration range, the amount of DCP present in solution is an indicator of the amount of template bound to the MIP. Competitive radioimmunoassays were used to determine the affinity of DCP for the 2,4-D-imprinted polymer and it was found that, while 500 pg of MIP were required to bind 50% of the CMMC, only 250 pg of polymer were required to bind 50% of the DCP. In order to verify this unexpected result, competitive binding assays were performed in the presence of C-2,4-D and it was found that the relative affinities of the polymer for CMMC and DCP were approximately 6 and 10% that of 2,4-D. However, while CMMC bound poorly to the non-imprinted polymer, DCP bound equally as well as to the MIP. This indicates that, in the latter case, binding is almost entirely non-specific. Thus DCP was a poor probe for this system. Fluorescein was also examined as a probe for polymers imprinted with... 

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