Enzymic Assays

Assays of ciguatoxin. Determination of ciguatoxin levels in fish was carried out in many laboratories by mouse assays. Enzyme immunoassay to screen inedible fish has been proposed by Hokama (9). No specific chemical assay has been developed, as information on functional groups suitable for fluorescence labeling is not available. Analyses conducted in the authors laboratory on remnant fish retrieved from patients meals indicated that ciguatoxin content as low level as 1 ppb could cause intoxication in adults. An extremely high sensitivity and a sophisticated pretreatment method will be required for designing a fluorometric determination method for the toxin. 

NOTE Cells were incubated for 18 hours in 250 jiM MDMA prior to assay. Enzyme activity was linear with respect to time under the culture conditions used, as shown at the right. Data presented as mean SEM. 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

Once solubilization of the membrane protein has been achieved, a reliable assay for it must exist. If the protein is an enzyme, then one must quantify the specific activity (spc. act.) of the enzyme, i.e., pmol product formed or substrate disappeared per min per mg protein. Thus, not only must the activity of the enzyme be assayed (44) but also the protein content of the enzymatic preparation. In this connection, Dashek and Micales (45) have discussed the factors that must be considered when assaying enzyme activity. In addition, they review protein quantification. 

An enzymatic method (Kll) of estimating galactose-l-phosphate in red blood cells is simpler and less time-consuming than the paper chromatographic method, though it requires more expensive reagents and apparatus. This method closely resembles methods of assaying enzyme activity and it is, therefore, described with them in Section 4.4.1. 

FIDAP software package, 7 25 Fidelity, of transmission signals, 11 131 Field assays, enzyme immunoassay kits for, 14 144 Fieldbus, 20 672 impact of, 20 673... 

With enzyme-multiplied immunoassay technique (EMIT) assays, enzyme tags are used instead of radiolabels. The antibody binding alters the enzyme characteristics,... 

Biomedical analytical chemistry happens to be one of the latest disciplines which essentially embraces the principles and techniques of both analytical chemistry and biochemistry. It has often been known as clinical chemistry . This particular aspect of analytical chemistry has gained significant cognizance in the recent past by virtue of certain important techniques being included very much within its scope of analysis, namely colorimetric assays, enzymic assays, radioimmunoassays and automated methods of clinical analysis. 

John RA. 1993. Photometric assays. Enzyme assays. A practical approach. Eisenthal R, Danson MJ, editors. Oxford Oxford University Press pp. 59-92. 

A method for the assay (enzyme aetivity) of carbonic anhydrase (Chap. 8. Zn(ll)) uses the catalysis at pH 7.0 of the hydrolysis reaction... 

Fig. 5.1 Principle of MS-based enzyme assays. Enzyme (E) molecules react with the substrate (S) to form an enzyme-substrate complex (ES), leading, for example, to a subsequent cleavage into two products Pi and P2. Pi and P2 are monitored continuously by ESI-MS. The injection of an inhibitor, I, results in the temporary formation of an inactive enzyme-inhibitor (El) complex, resulting in a reduction of Pi and P2 and negative peaks in the corresponding mass traces.

Assay Enzyme Enzyme pEGFR pErbB2 pErbB2 pErbB2 Growth... 

Use of a surrogate end point that is quick and easy to obtain Permeation experiments using a radiolabeled, fluorescent, HPLC-detectable, or radio immuno assay/enzyme linked immuno sorbent assay-detectable marker necessitate the need of extensive sample handling and sample analysis. This accentuates the cost of sample analysis and overall time spent in characterizing the efficacy of formulations. Furthermore, current state of the art fluidics systems put a fundamental limit on the number of samples handled in a given time. 

Enzyme-linked immunosorbent assays. An indirect application of enzymes in analysis is as a marker or label in enzyme-linked immunosorbent assays (ELISA). In ELISA, the enzyme does not react with the analyte instead, an antibody is raised against the analyte (antigen or hapten) and labelled with easily assayed enzyme, usually a phosphatase or a peroxidase. The enzyme activity is proportional to the amount of antibody in the system, which in turn is proportional, directly or indirectly depending on the arrangement used, to the amount of antigen present (Morris and Clifford, 1984). 

It is extremely common for intermediates to occur after the initial enzyme-substrate complex, as in equation 3.19. However, it is often found for physiological substrates that these intermediates do not accumulate and that the slow step in equation 3.19 is k2. (A theoretical reason for this is discussed in Chapter 12, where examples are given.) Under these conditions, KM is equal to Ks, the dissociation constant, and the original Michaelis-Menten mechanism is obeyed to all intents and purposes. The opposite occurs in many laboratory experiments. The enzyme kineticist often uses synthetic, highly reactive substrates to assay enzymes, and covalent intermediates frequently accumulate. 

In this assay, enzymes with chymotrypsin-like specificity readily bind the side chain of Phe at the primary substrate binding site (SI) and subsequently hydrolyze the adjacent amide bond linking the Phe residue to the p-NA moiety. On cleavage, the release of p-NA is measured by the increase in absorbance at 410 nm (e4l0 = 8480 M 1 cm-1) with time, using a recording spectrophotometer. 

Vorderwiilbecke, T., Kieslich, K., and Erdmann, H. 1992. Comparison of lipases by different assays. Enzyme Microbiol. Technol. 14 631-639. 

Wall, D. B., Finch, J. W., and Cohen, S. A. (2004). Comparison of desorption/ionization on silicon (DIOS) time-of-flight and liquid chromatography/tandem mass spectrometry for assaying enzyme-inhibition reactions. Rapid Commun. Mass Spectrom. 18 1482-1486. 

Routine qualitative and quantitative biochemical analysis including many colorimetric assays. Enzyme assays, kinetic studies, and difference spectra. 

Michaelis-Menten and Lineweaver-Burk plots can help classify an inhibitor, but successful drug development requires the ability to compare the effectiveness of one inhibitor to another based on results from an assay. Enzymes are typically compared based on one of two values K or IC5(). 

The EMMIA system was developed by Ngo and Lenhoff (N3, N4). In this assay, enzyme activity is modulated by an enzyme modulator which is coupled to antigen (free form) but not by the complex of enzyme modulator-antigen and antibody (bound form). As shown in Fig. 2 and Table 6, in an enzyme inhibitor immunoassay, an enzyme inhibitor is used as a negative modulator. For example, the reaction mixture for measuring thyroxine consists of acetylcholine inhibitor-thyroxine conjugate [I-Ag], acetylcholinesterase [E], unlabeled thyroxine [Ag], and antithyroxine antibody [Ab]. When the amount of unlabeled thyroxine, which binds to antibody [Ab Ag], is increased, the free form of acetylcholine inhibitor-thyroxine conjugate [I-Ag] increases, and the enzyme activity decreases. Therefore, the enzyme activity is inversely proportional to the concentration of unlabeled thyroxine. A tetrazyme kit (Abbott) is now available for measuring thyroxine. 

Table 5.2 Commonly Assayed Enzymes for Organ- and Disease-Specific Diagnoses...

Immunosorbent assays (enzyme-linked or fluorescent-linked, cell signaling, PathScan)... 

The development of the HPLC method to assay enzyme activities has made it considerably easier to assay a single activity in the presence of others. Thus, attempts to obtain a pure protein during the purification procedure may not be necessary. Since the advent of HPLC to assay enzyme activities, it is possible to stop the purification at a much earlier stage and still assay for a single enzymatic activity. In fact, for some studies, it is even advantageous to assay the activity of interest in the presence of other activities. 

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