Enzyme assay fluorescence

See also Bioluminescence. Chemiluminescence Liquid-Phase. Electrophoresis Blotting Techniques. Enzymes Immobilized Enzymes Enzyme Assays. Fluorescence Quantitative Analysis. Forensic Sciences Blood Analysis. Immunoassays Overview. Immunoassays, Applications Clinical Forensic. Immunoassays, Techniques Radioimmunoassays. 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Hybrid probe—immunoassays are expected to find a specific niche in clinical analysis, especially as a means to adapt probe assays to existing immunoanaly2ers which are locked into a specific enzyme or fluorescence detection technology. Commercialization of the first of these assays is expected by the year 2000. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

Note All NAD+- and NADP - linked enzyme assays are also capable of being monitored fluorimetrically. The fluorescent compounds in each assay are shown in bold type. ... 

A protocol for continuous enzyme assay that involves one or more auxiliary enzymes to convert a product of the primary reaction in a second or auxiliary reaction that produces a change in absorbance or fluorescence. As noted below, coupled enzyme assays, while convenient, are fraught with experimental limitations that must be overcome in order to obtain valid initial velocity data. 

Coumarins also have a C6-C3 skeleton, but they possess an oxygen heterocycle as part of the C3-unit. There are numerous coumarins, many of which play a role in disease and pest resistance, as well as UV-tolerance. The coumarin umbelliferone (1.21) is popular in enzyme assays. Umbelliferone esters can be used as a substrate for non-specific esterase enzyme assays and in fluorescent immunoassays (Jacks and Kircher, 1967). In order to quantify the enzyme activity of the popular reporter gene P-glucuronidase (GUS), plant extracts can be incubated with 4-methylumbelliferyl P-D-glucuronide (4-MUG 1.22), which upon hydrolysis... 

P 75] A static enzyme assay experiment was carried out using a stopped-flow method [161]. This is commonly used for monitoring reaction kinetics. P-Galacto-sidase was used as model enzyme to convert the substrate fluorescein mono-p-D-galactopyranoside (FMG) via hydrolysis into fluorescein. As buffer solution 10 mM potassium phosphate at pH 7.2 with 1 mM ascorbic acid was used to minimize photobleaching. The enzymatic reaction is accompanied by a change in fluorescence intensity which can be monitored with a microscope. 

Analysis is best carried out by a fluorescence activated cell sorter (see 10.7.5) but, if the cells are pulse labelled with [3H]-thymidine immediately before harvesting the proportion of cells in S-phase in the various fractions can be estimated by autoradiography (see 12.3). The problem with this procedure is that the machines can become contaminated with radioactivity and the tritium may interfere with subsequent enzyme assays. Labelling of a sample after fractionation is a poor alternative, but prior pulse labelling with bromodeoxyuridine allows S-phase cells to be detected using a fluorescent antibody 12.7.5. 

C, showing its maximal activity above 95°C and thermostability with a t1/2 of 85 h at 75 °C. Using a special stainless steel optical pressure cell, enzyme assays and fluorescence measurements up to pressure of 160 atmosphere boiling the sample have been performed (D Auria et al., 1999). The enzyme showed maximal activity at 125 °C. 

Fluorescence assays for biotransformations are an indispensable tool for enzyme engineering and the daily practice of enzyme studies. Fiuorogenic substrates are particularly useful as general probes for enzyme classes that can be used in routine screening and activity checking. However, they cannot replace the authentic substrate in cases where an optimization towards a particular biotransformation is desired. In such cases an indirect fiuorogenic assay or an instrumental assay may be required in order to follow the reaction. A variety of fluorescence assays for enzymes still remain to be discovered and the development of new enzyme assays... 

Luminescent RE + chelates have been successfully developed as labels and probes for highly sensitive and selective bioassays in the past two decades. Time-resolved Inmines-cence detection has been widely applied in fluoroimmunoassay, DNA hybridization assay, enzyme assay, cell activity assay and fluorescence imaging microscopy. ... 

Sohm et al. (in press) have extended their studies of APA and P uptake kinetics by Trichodesmium colonies in the N. Atlantic to a comparative study of these two indices in the tropical N. Pacific and coastal northern Australian waters as well. They detected sharp contrasts among the sites with much higher Riax and APA values in their tropical N. Atlantic stations compared to the N. Pacific and northern AustraHa indicating more severe P hmitation in the tropical N. Atlantic. Dyhrman et al. (2002) also reported evidence of severe P stress in Trichodesmium in the N. Atlantic based on a enzyme linked fluorescence (ELF) assay. A follow up study also saw less evidence of P stress in samples from the N. Pacific (Hynes et al., Pers. Comm.). 

Nonradiometric methods iticlude absorbance, fluorescence. and luminescence spectroscopy. Enzyme assays are a common example. The as.suy is usually run at or below the value of thq substrate, with only about S% of the substrate consumed during the assay, and multiple enzyme turnovers occur during the a.ssay. Sometimes enzyme reactions arc coupled, especially if the target reaction does not produce a product that can be detected directly in the assay. An example is carboxypeptidase. which is coupled to the teduction of NADP to NADPH. giving ri.se to absorbance at 340 nm. 

The fluorescence-based assay described herein is used to screen large libraries of compounds, in 96-well format, for the ability to inhibit HIV protease and to accurately determine the affinity of identified inhibitors for the enzyme. Much of the discussion in this section will be widely applicable to fluorescence-based assays and enzyme assays in general. Because numerous potent inhibitors of HIV protease have been identified, part of this procedure outlines the analysis required to deal with these potent compounds. Like any enzyme assay, preparing this assay for routine use can be divided into three parts (1) determination of an appropriate enzyme concentration for assay, (2) determination of the substrate concentration dependence (Km and Vmax), and (3) determination of inhibitor concentration dependence (IC50 and K, values). Parts one and two do not need to be repeated every time inhibitor assays are run—only once to check new batches of enzyme or to troubleshoot any problems with the assay. 

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