Comparison with enzyme assays

Chou C., Tsai Y, Liu J., Wei J.C.C., Liao T., Chen M., and Liu L. 2001. The detection of the HLA-B27 antigen by immunomagnetic separation and enzyme-Unked immunosorbent assay—Comparison with a flow cytometric procedure. lournal of Immunological Methods 255 15-22. 

In cases where the mode of action is the strong or irreversible inhibition of an enzyme system, the assay may measure the extent of inhibition of this enzyme. This may be accomplished by first measuring the activity of the inhibited enzyme and then making comparison with the uninhibited enzyme. This practice is followed when studying acetylcholinesterase inhibition by organophosphates (OP). Acetylcholinesterase activity is measured in a sample of tissue of brain from an animal that has been exposed to an OP. Activity is measured in the same way in tissue samples from untreated controls of the same species, sex, age, etc. Comparison is then made between the two activity measurements, and the percentage inhibition is estimated. 

Many more papers deal with rhizosphere phosphatase activity (63-83) in the presence of a number of different plant species this will partly be due to the simplicity of the enzyme activity assay (85,86) and the generally reported, well-correlated variation trends among organic and inorganic phosphorus content and phosphatase activity. More precisely, closer to the roots, the inorganic P depletion zone in comparison with bulk soil is more pronounced in addition, organic and inorganic P contents are inversely correlated, and the mineralization rate of or-... 

Figure 3(A). Comparison of temperature optima for activities of glucose isomerase, amylase, and >galactosidase. Enzymes were assayed with cell extract from xylose-grown cells. A 100% activity value corresponds to 0.60, 0.58, and 0.46 U/mg for glucose isomerase, amylase, and -galactosidase, respectively. Cell extracts in 50 mM sodium phosphate buffer (pH 7.0), 100 mM sodium acetate buffer (pH 5.5), and 100 mM sodium phosphate buffer (pH 6.0) for glucose isomerase, amylase, and -galactosidase, respectively, were preincubatcd at the indicated temperatures, prior to the assay for residual enzyme activities. Reprinted with permission from ref. 20. Copyright 1990 American Society for Microbiology.

Even under seemingly ideal conditions, the steady-state concentration of the first reaction product may exceed the inhibitory constant for the binding of that product to the primary enzyme. In such cases, the linearity of the coupled assay can be misleading, and the investigator must validate the coupled enzyme kinetic data by direct comparison with the results obtained by another technique such as a stopped-time radiometric assay. This... 

The simplest, but least accurate, method of assaying DPO activity is to record the final color yield when the enzyme is incubated with a suitable chromogenic substrate such as catechol, DOPA, or 4-methylcatechol. DOPA is the most frequently used substrate in colorimetric assays because it yields a dark brown/black end-product. In this reaction, catecholase catalyzes the conversion of DOPA to dopaquinone and then to the red dopachrome, which subsequently polymerizes to yield dark brown melanin-type pigments. Unfortunately, this simple procedure has serious limitations, as it measures the end-product of a sequence of reactions rather than the true initial reaction rate. Furthermore, because different substrates yield different final colors, valid kinetic comparisons between substrates are not possible. Nevertheless, this simple assay technique has proved adequate for useful comparative studies of the levels of enzymic browning in different fruit varieties and similar problems (Vamos-Vigyazo, 1981 Machiex et al., 1990). 

Nunes, G.S., M.P. Marco, M. Farre, et al. 1999. Direct application of an enzyme-linked immunosorbent assay method for carbaryl determination in fruits and vegetables. Comparison with a liquid chromatography-postcolumn reaction fluorescence detection method. Anal. Chim. Acta 387 245-253. 

Positive control inhibitors for each of the major CYP enzymes should also be included to further demonstrate that the test system is performing as expected. The direct-acting inhibitors used in our laboratory are summarized in Table 7, along with the IC50 values determined during assay validation and a comparison with literature values. It is worth noting that the positive control inhibitors used for CYP inhibition studies need not necessarily be CYP-selective inhibitors, in contrast to those used for reaction phenotyping, which should be CYP-selective inhibitors. 

To illustrate the rapidity of HPLC, particularly in comparison with the more conventional techniques, the same sample was separated by conventional ion-exchange chromatography. Figure 5.10 compares the two procedures. These data show that where 14 hours was required for the traditional method, only about 45 minutes is required with HPLC. Therefore, the total time needed to carry out this purification, not counting the time for the enzyme assay, could be as short as 3 to 4 hours. If necessary, the chromatography step could be completely automated. Finally, since each run will use only a fraction of the total volume of the starting material, the entire procedure will be economical. 

Enzyme DNA hybridization assays with electrochemical detection can offer enhanced sensitivity and reduced instrumentation costs in comparison with their optical counterparts. Efforts to prevent non-specific binding of the codissolved enzyme and to avoid fouling problems by selecting conditions suitable to amplify the electrode response have been reported by Heller and co-workers [107]. A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode was described by Limoges and co-workers for an enzyme nucleic acid hybridization assay using alkaline phosphatase [108] or horseradish peroxidase [109]. In another methodology to improve sensitivity, a carbon paste electrode with an immobilized nucleotide on the electrode surface and methylene blue as hybridization indicator was coupled, by Mascini and co-workers [110], with PGR amplification of DNA extracted from human blood for the electrochemical detection of virus. 

An enzyme linked immunosorbent assay (ELISA) has been developed and applied to assay of amoxicillin in lung secretions in comparison with conventional bioassay [182]. The ELISA was capable of measuring amoxicillin down to 10 ng/ml, but it also responded to amoxicillin penicilloic acid and consequently gave results higher than bioassay. A commercially available ELISA system was reported to have a sensitivity of 6 ng/ml for amoxicillin in milk [183],... 

The classic quantitative biochemical method for assaying steroid receptors in tumor tissue specimens is the multiple-point dextran-coated charcoal (DCC) titration assay. However, in comparison with the classic DCC assays, enzyme immunoassays are preferred as they cost less and are simpler, require less time, and can be performed using less tissue than DCC titration assays. 

At any given concentration, the initial velocity rate (Vg) is proportional to the enzyme concentration so the term Vg can be used as a measure of the enzyme concentration provided certain precautions are taken. One should be aware of the variability of these reaction constants with pH, temperature, and ionic strength. Likewise, conditions for substrate concentration should be selected to guarantee no interference with the reaction rate. Usually, the concentration of the substrate is chosen to be well above the value to ensure acceptable conditions (H3). As a side issue, it should be noted that substrates with a free N-terminal are 10 to 100 times more soluble than the corresponding benzoyl derivatives CTIO). Finally, one should always report the conditions under which the assay was run to allow comparison with other laboratories. 

There is a vast and continually growing literature on these two enzymes, but many papers have not distinguished clearly between them. The purpose of the present survey is to focus attention on human serum cholinesterase and its variants in the light of recent research, and to offer a critical assessment of reports of its physical and chemical properties and clinical and toxicological applications. The methodologies of cholinesterase assays and the older literature on the variant enzymes will not be considered in detail Relevant references may be found in several authoritative reviews (B15, D5, F4, G13, G16, H5, K5, LIO, L24, Ul, W35). Acetylcholinesterase, also, has been extensively reviewed (Nl, S22, S23, W29) and it will be discussed here only in order to make particular points of comparison with cholinesterase. 

In 1960, at the general assembly of the International Pharmaceutical Federation (FIP), the obsolescence of various national pharmacopeial methods for assaying pharmaceutical enzymes was demonstrated. An international commission on pharmaceutical enzymes was created to deal with this unsatisfactory situation and develop improved assay methods and guidelines for the preparation of pharmaceutical enzyme reference materials. The Center for Standards has a coordination function in organizing collaborative enzyme assays between academic, industrial, and national pharmaceutical control laboratories and in distributing FIP pharmaceutical enzyme standards. Since 1960, many FIP assay methods and standard preparations have been adopted by national and international pharmacopeias, such as the European Pharmacopoeia. The ultimate goal is to provide official, preferentially nonempirical, standardized assay methods by which comparison of commercially available pharmaceutical enzymes is made possible. The most desirable situation would be an international uniformity of enzyme standards and assay methods, which would allow physicians and clinicians to unambiguously compare the potencies of commercially available enzyme products. 

J Grenier. Plasma estradiol-17-beta assay, using an enzyme immunoassay kit— Comparison with a specific radioimmunoassay. J Steroid Biochem 33 833, 1989. 

---