Competitive chemiluminescence assay

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 

propranolol, atrazine and theophylline but, just as in our work (Section 20.2.6.2.) and that of Piletsky et al. (Section 20.3.1.), structurally unrelated probes are generally unsuitable. Incidentally, Haupt and co-workers have successfully used 

5- dihydroxyphenylacetic acid (homogentisic acid) as a reporter in a 2,4-D MIP-based sensor using differential pulse voltammetry [52]. 

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A chemiluminescent assay using competitive immunological techniques can be easily designed when chemiluminescent antigens are employed. In this case chemiluminescent antigen and sample compete for immobilized antibodies. Depending on the nature of the reaction antigen, bound and unbound label can also be separated by adsorption onto charcoal. 

Chemiluminescent labels may be employed in sandwich or competitive antigen assays. In sandwich assays, a solid support holds a primary antibody, and incubation with ligand yields a species that is detectable following a second incubation step with a labeled second antibody. Luminol has been tested as an immunoassay label it may be coupled to proteins through its primary amino group. Luminol reacts with hydrogen peroxide and hydroxide in a microperoxidase-catalyzed reaction, which yields light at 430 nm (Eq. 6.8) ... 

The normal reference interval for TPOAb is controversial. With sensitive assays, low concentrations of TPOAb may be detected in some healthy individuals without thyroid disease. There is a high prevalence of anti-TPO antibodies in the elderly. However, longitudinal studies suggest that the presence of TPOAb is a risk factor for autoimmune thyroid dysfimetion. TPO antibody concentrations are usually expressed as units per milliliter with reference to the MRC Standard 66/387. With a competitive IRMA, the mean TPOAb activity in normal sera is 69 15 U/mL (SEM). Using a sensitive chemiluminescence assay, values are less than 2 U/mL. ... 

CLIA is similar to EIA and ELISA techniques except that the final receptor enzyme assay is replaced with a chemiluminescent tracer followed by measurement of light released as a result of the chemical reaction. The principles of a chemiluminescence competitive binding assay are shown in Figure 8-8. 

Competitive binding chemiluminescence assay. The chemiluminescent competitve binding assay is similar to a RIA. The acridinium ester-labeled substance and the endogenous substance compete with the specific antibody that is available in limited concentration. The concentration is determined by the quantity of tracer that binds to the antibody. The use of solid-phase antibody simplifies separation of free from bound tracer. 

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

We have developed chemiluminescent immunoenzymatic assays for (3-ago-nist drugs in the 96-well-microtiter-plate format. Such competitive assays have been used for determination of clenbuterol and of the overall content of p-agonist drugs in the sample. They matched the standard requirements of precision and accuracy, and were more sensitive compared to the conventional colorimetric methods. Moreover, CL detection was very rapid, making these assays suitable for screening analysis. 

To test our new signal reagent based on GZ-11 the detection system was applied to two competitive-format immunoassays. These two assays (for atrazine and clenbuterol) normally use chromogenic detection systems. While these colorimetric assays may be adequate for laboratory use, chemiluminescent detection offers potential advantages in sensitivity and on site screening applications [33],... 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

The availability of MIP microparticles through this synthetic method has also stimulated the development of analytical techniques that make use of them as sensing elements. Apart from competitive radioassays [30] and immunoassays [32], which were already performed with ground bulk polymers, the small, regular size of the beads prepared by dispersion/precipitation polymerisation enables their use in CEC [45, 46], scintillation proximity assays [35], fluorescent polarisation assays [47], and chemiluminescence imaging [48]. 

Chemiluminescent labels may also be used in labeled-antigen (competitive) assays. The antigen (analyte) competes with the labeled analyte for immobilized antibody, and, following a rinse step, reagents are added to generate chemiluminescence from the labels. 

The analytical detection limits of competitive and noncompetitive immunoassays are determined principally by the affinity of the antibody and the detection limit of the label used, respectively. Calculations have indicated that a lower limit of detection of lOfmol/L (Le., 600,000 molecules of analyte in a typical sample volume of 100 jiL) is possible in a competitive assay using an antibody with an affinity of iO L/mol. Table 9-2 illustrates the detection limits for isotopic and nonisotopic labels. A radioactive label, such as l, has low specific activity (7.5 million labels necessary for detection of 1 disintegration/s) compared with enzyme labels and chemiluminescent and fluorescent labels. Enzyme labels provide an amplification (each enzyme label produces many detectable product molecules), and the detection limit for an enzyme can be improved by replacing the conventional photometric detection reaction by a chemiluminescent or bioluminescent reaction. The combination of amplification and an ultrasensitive detection reaction makes noncompetitive chemiluminescent EIAs among the most sensitive types of immunoassay. Fluorescent labels also have... 

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