Colorimetry enzyme assay

The method used for detection depends on the type of label used. Isotopic counting is employed for radioisotopes, colorimetry for enzyme assays, luminescence and fluorescence measurements can be achieved by means of photomultiplier tubes, while turbidimetry or nephelometry is used for particle enhanced assays. 

Two publications on the higher ketose mono- and bis-phosphates in rat-liver extracts have described their detection and estimation by colorimetry and enzymic assay,and the synthesis of octulose 1,8-bisphosphates using muscle aldolase. A stereospecific synthesis of adenosine 3, 5 -cyclic phosphothioate has appeared/ Standard condensation methods have been used to synthesize the 5 -O-(Taminoethane-phosphonyl) nucleosides (21) and (22). The compounds were shown to be inert to the action of alkaline phosphatase and are poor substrates for 5 -nucleotidase. The selenophosphates (23) and (24) have been prepared.Phosphorylation of nucleosides using dibenzyl hydrogen phosphate,... 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

Early work demonstrated the use of enzymes coupled to antibodies or antigens as reagents in immunoassay. Enzyme activity can be measured in a variety of ways, each with certain advantages, which makes a variety of enzymes good labeling substances. Most assay methods are based on spectroscopic properties derived from an enzymatically transformed substrate. These methods are colorimetry, flurorometry, luminometry, and electrometry. 

False negative results for cTn may occur if the assay is insufficiently sensitive, especially in enzyme-linked immunosorbent assay (ELISA) methods where colorimetry rather than fluorescence or chemiluminescence is used as the detection method, and in those assays with poor cross-species reactivity. False negative results may also occur in samples that have deteriorated. Serum cTn is stable at -70°C, but it deteriorates several percent per day at 4°C and by 10% per week at -20°C. Rare false positive results may occur with circulating heterophilic antibodies, fibrin clots, or incomplete serum separation (or in the presence of rheumatoid factor). Hemolysis produces negligible interference. 

Figure 4.14 Polymer brushes with enzymatic activity. (A) Enzymes are physisorbed in polymer brushes with different character (e.g, neutral or charged). (B) The activity of the enzymes such as alkaline phosphatase and horseradish peroxidase (rightj is assayed with colorimetry.

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