Enzymes colorimetric detection

Parasite Antlbodies-containing Conjugated enzyme Colorimetric detection... 

CFA with ISE detection has been applied to determinations with enzyme electrodes [81]. It has been shown [115] that potentiometric detection with ISEs is superior to colorimetric detection in its simpler methodology, high selectivity, a wide linear dynamic range, and insensitivity to solution colouration... 

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

Cheng Q, Stevens RC. Coupling of an induced fit enzyme to polydiacetylene thin films colorimetric detection of glucose. Adv Mater 1997 9 481-483. 

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-amino-phenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05-1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. 

To improve the quality control process of gasohol and hydrated ethanol, an automated FIA system was developed using AOD and HRP enzymes, and addition of 4-aminophenazone and phenol. A colorimetric detection method was used in two different methods of analysis, with free (4) and immobilized enzymes. Both systems have shown good results when compared with established methods such as gas chromatography (GC) and high-performance liquid chromatography (HPLC) (4,7). 

In the selected example by Lam et al. [101] many peptide libraries were prepared using the mix and split technique and tested in different on-bead screens. Incomplete libraries were tested (the population of most of them was more than a million compounds), and the positive structures were exploited through focused libraries. Some libraries were screened against an anti-insulin monoclonal antibody tagged with alkaline phosphatase, which allowed an enzyme-linked colorimetric detection. Only the beads bound to the murine MAb showed a tourquoise color, while the vast majority remained colorless (details of the technical realization of the assay can be found elsewhere [101, 102]). The chemical structure linked to the positive beads was then easily determined via Edman degradation of the peptide sequences. 

It should be mentioned that most natural aldolase enzymes can also be assayed using enzyme-coupled systems relaying the reaction to a redox process with NAD. The formation of NADH by active microbial colonies in expression libraries of mutant enzymes was detected colorimetrically in agar plates using phenazine methosulfate and nitroblue tetrazolium, which forms an insoluble precipitate. The assay was used by Williams et al. [14] and Woodhall et al. [15] for evolving sialic acid aldolases to accept non-natural aldehyde acceptors. 

Again, there are several methods for measuring individual phospholipids enzymatic assays for total phospholipids provide a convenient starting point. Because most plasma phospholipids contain choline, the enzyme phospholipase-D is used to release choline from phospholipids. The choline is then oxidized by added choline oxidase to yield betaine and hydrogen peroxide, which are then measured using a peroxidase-linked colorimetric detection method. In cases of phospholipidosis, it is essential to use cells (e.g., leukocytes) to demonstrate the accumulation of phospholipids. 

One prominent use of organometallic complexes is in metallo-immuno assays. The traditional radio-linked immuno assay (RIA) is highly sensitive but has obvious disadvantages related to the use of radioactivity. Modern alternatives use colorimetric, fluorescence, or enzyme-linked detection schemes (ELISA). The idea of using non-radioactive metals for specific, highly sensitive detection in immuno assays was first mentioned by Gais, who used steroid... 

Chapters 17-22 describe the hybridization of the nonradioactive probes to the DNA and RNA immobilized on blots, together with the detection systems necessary to reveal where the probe has hybridized. Chapters 17-19 deal with digoxigenin probes, with Chapters 17 and 19 describing chemiluminescent detection on DNA and RNA blots respectively, and Chapter 18 describing a colorimetric detection system. Chapter 20 deals with enhanced chemiluminescent detection of enzymically labeled probes, whereas Chapters 21 and 22 describe enhanced chemiluminescent detection of large (Chapter 21) and small (oligonucleotide. Chapter 22) probes labeled with fluorescein. 

Colorimetric detection is the most commonly used method for the detection of enzyme activity. Fluoro-metric or chemiluminometric assays are also utilized when high sensitivity is desired because they are often more than 10-fold more sensitive. In fact, fluo-rometric or chemiluminometric assays can be several thousand times more sensitive when using jS-D-gal-actosidase or alkaline phosphatase as the conjugating enzyme (Table 1). 

The Chemical Agent Water Test Kit M272 consists of colorimetric detector tubes and enzyme impregnated detection tickets for nerve agent detection. The kit is designed to detect the presence of chemical agents in water samples. 

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