Enzymes competitive

Table 3.2 Some examples of competitive enzyme inhibitors in clinical use...

Figure 7.6 Double reciprocal plot for a tight binding competitive enzyme inhibitor, demonstrating the curvature of such plots. The dashed lines represent an attempt to fit the data at lower substrate concentrations to linear equations. This highlights how double reciprocal plots for tight binding inhibitors can be misleading, especially when data are collected only over a limited range of substrate concentrations.

Figure 2 Schematic picture of a competitive enzyme-linked immunosorbent assay (ELISA).

Several qualitative and quantitative immunochemical methods for CAP analysis in biological matrices of animal origin have been described [101,102, 104,105] (see Table 3). Van de Water et al. [ 102] described an ELISA that detected CAP in swine muscle tissue with an IC50 value of 3 ng mL1. This immunoassay was improved and subsequently optimized incorporating the streptavidin-biotin amplification system. There are also several commercially available test kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter plate. The measurement is made photometrically, obtaining a LOD of 100 ng L 1 in meat and eggs and 150 ng L 1 in milk. The test has been also applied to the analysis of tetracyclines. 

On the other hand, the 5091 CAP lp test is a direct competitive enzyme immunoassay for the quantitative analysis of these residues in milk, eggs, meat,... 

Competitive enzyme inhibition, 10 256 Competitive intelligence work, 15 645 Competitiveness, organizational,... 

Neal JM, Kunze KL, Levy RH, et al. Kiiv, an in vivo parameter for predicting the magnitude of a drug interaction arising from competitive enzyme inhibition. Drug Metab Dispos 2003 31(8) 1043-1048. 

Certain drugs such as chloramphenicol require additional tests for their detection and quantification in meat tissues. The Competitive Enzyme Labeled Immunoassay for Chloramphenicol (CELIA) was developed and is used by FSIS laboratories to detect and quantify this drug in the meat supply chloramphenicol is not approved for use in food animals. CELIA detects 5 ppb chloramphenicol in tissue extracts. 

Schnitzius, J. M., Hill, N. S., Thompson, C. S. and Craig, A. M. 2001. Semi-quantitative determination of ergot alkaloids in seed, straw, and digested samples using a competitive enzyme-linked immunosorbent assay. Journal of Veterinary Diagnostic Investigation, 13 230-237. 

Bober, M. A., Kurth, M. J., Milco, L. A., Roseman, D. M., Miller, R. B. and Segal, H. J. 1991. A pyrrolizidine alkaloid-enzyme-linked immunosorbent-assay detection strategy. ACS Symposium Series, 451 176-183 and, Bober, M. A., Milco, L. A., Miller, R. B., Mount, M., Wicks, B. and Kurth, M. J. 1989. A competitive enzyme-linked immunosorbent-assay (ELISA) to detect retronecine and monocrotaline in vitro. Toxicon, 27(9) 1059-1064. 

As classical competitive enzyme inhibitors, they have a high affinity for the active site but are not substrates. The enzyme is occupied by the inhibitor for relatively long periods, and therefore cannot handle ACh efficiently, as a result of the saturation phenomenon. 

Figure 8.3. Schematic representation of a non-competitive enzyme-linked immunosorbent assay using the sandwich technique.

Indirect competitive enzyme-linked immunosorbent assay... 

Sulfadiazine Swine bile and Direct assay Direct competitive enzyme-linked 32-36 67... 

Milk Acidification, centrgn, pH Direct competitive enzyme-linked 1 178... 

Swine liver and Matrix solid-phase Direct competitive enzyme-linked 5 59... 

Milk Direct assay Indirect competitive enzyme- 0.05 66... 

Sulfathiazole Honey H2O diln Indirect competitive enzyme- 300 179... 

A competitive enzyme immunoassay for the quantification of ivermectin residues in bovine liver has also been reported recently (85). This method uses a polyclonal antiserum raised in rabbits against 5-O-succinoylivermectin-trans-ferrin conjugate. Cross-reactivity was demonstrated with doramectin, a member... 

During in vivo studies under biologically relevant conditions, the cis-Pt loading of the DNA is much lower than for the above-mentioned in vitro studies. It has been calculated that mortality of HeLa cells occurs at an value of 10 5 (i.e., one bound cis-Pt molecule per 105 nucleotides) (64a). This excludes atomic absorption spectroscopy for identification of the in vivo adducts. Immunochemical techniques, however, have shown to be very promising, and high sensitivity and selectivity levels have been reached. At the moment, only a few studies in which antibodies are raised against cis-Pt-treated DNA (64) or against synthetic cis-Pt adducts with mono- or dinucleotides are available (64a). With the latter method, quantitation of the different platinum-DNA adducts formed under in vivo conditions is possible. At the moment, femtomole (10-15 mol) amounts of the adducts can be detected with competitive enzyme-linked immunosorbent assay (ELISA) techniques. It has been demonstrated in this manner that the GG-Pt adduct is also the predominant adduct under in vivo conditions. 

At their most elaborate, epitope mapping techniques can provide detailed information on the amino acid residues in a protein antigen, which are in direct contact with the antibody binding site. X-ray crystallography of antibody-antigen complexes can identify contact residues directly and unequivocally, though not surprisingly in view of the effort required, this method is not in routine use. At the other extreme, demonstration by competition enzyme-linked immunosorbent assay (ELISA) methods that two antibodies bind to different sites on... 

Fig. 9.12. High-throughput screening of enantioselective catalysts by competitive enzyme immunoassays [ 18]. The solid antibody recognizes both enantiomers, and tire hatched antibody is (S)-specific, enabling die determination of yield and ee.

Several case reports have suggested that the elimination of ciclosporin can be impaired by oral contraceptives, resulting in increased plasma ciclosporin concentrations. Ciclosporin undergoes hydroxylation and /V-dcmethyla-tion, in which cytochrome P450 is involved, so competitive enzyme inhibition probably explains the interaction. Dosages of ciclosporin should be reviewed carefully (340). 

HIV protease inhibitors are transition-state analogs (Chapter 9, Section 9.2.6) HIV protease binds them much more tightly than the natural substrate because the substrate must be distorted to assume its transition-state configuration. Thus, HIV protease inhibitors are competitive enzyme inhibitors and thereby prevent the maturation and infectivity of the viral particle. They offer advantages over RTIs with respect to efficacy, safety, and occurrence of resistance. However, just like RTIs, HIV protease inhibitors are most often offered in combination with therapies based on another mode of action. Structures and data for the five approved HIV protease inhibitors are shown in Figure 13.14 and in Table 13.5. 

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