Chain Colorimetric assay

To enhance the applicability of UV spectrophotometry in pharmaceutical steroid analysis by increasing its selectivity and sensitivity, various methods have been developed based on chemical reactions leading to colored derivatives. Although their importance has decreased considerably, some of these methods are still in use in pharmacopoeias, mainly in the assay of formulations. For example, the A" -3-oxo and A " -3-oxo steroids can be determined as iso-nicotinoyl hydrazones (/Uax 380 and 410 nm 11500 and 17000, respectively). The dihydroxy-acetone side chain of corticosteroids at C17 reduces tetrazolium reagents to colored formazans (Amax 485 and 525 nm with triphenyl tetrazolium chloride and Tetrazolium Blue reagents, respectively e 16000 and 24000), thus creating the basis for a stability-indicating indirect colorimetric assay. Corticosteroids... 

Colorimetric procedures used In steroid assays are often subject to drug Interference. In the determination of 17-Ketosterolds by the Zimmerman reaction, drugs with the 17-Keto basic structure such as ascorbic acid, morphine and reserplne will cause Increased values. In the determination of 17,21 -dlhydroxysterolds by the Porter-Sllber reaction the dlhydroxy-acetone chain Is the reactive unit. Drugs like meprobamate, chloral hydrate, chloropromazlne and potassium Iodide will Interfere with this reaction and cause elevated values. In the colorimetric determination of vanlllylmandellc acid (VMA) by a dlazo reaction, drugs like methocarbamol and methyl dopa cause... 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

The on-bead assay was conducted according to Scheme 3.19, which shows the chain of events, which leads to a colorimetric response, when an oligosaccharide binds effectively to the B. purpurea lectin. The lectin was covalently linked to biotin, a small molecule with an extremely high affinity for streptavidin. The bead-lectin-biotin conjugates were then exposed to streptavidin, linked to the enzyme alkaline phosphatase. Alkaline phosphatase hydrolyses phosphate esters [e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 110]. When the 5-bromo-4-chloro-3-hydroxyindole (111) is released, in the presence of nitro blue tetrazolium (NBT), it forms a dark purple, insoluble dye, thus staining beads where there was a favorable binding interaction. 

Methyl Esters. These long-chain acyl esters are subjected to thin-layer chromatography, colorimetric analysis, and gas-liquid chromatography, coupled with mass spectrometry, as described in Chapter 4. These assays will provide information on the ester/P molar ratio (based on starting phosphate value it should be 2.0) and on the composition and relative distribution of fatty acyl residues. 

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

Rat adipose tissue homogenate has been incubated with cyclic AMP, ATP, and magnesium chloride in potassium phosphate buffer, pH 6.7, and the reaction terminated by pipetting aliquots of the mixture into chloroform plus potassium phosphate buffer, pH 6.8, for extraction of free fatty acids and assay by the method of Duncombe [93,169]. The latter is a colorimetric procedure for the determination of long-chain fatty acids in the range of 0.05 to 0.5 jiimol. 

The autoxidation of hydroxylamine to nitrite also involves a radical chain process (Kono, 1978), and the reaction is carried out at high pH. The assay was originally utilized by Elstner and Heupel (1976) who initiated the autoxidation by O2 generated by the xanthine/xanthine oxidase reaction. Nitrite formation was determined colorimetrically at 530 nm by diazo coupling with a-naphthylamine and superoxide dismutase was found to inhibit end product formation. Kono (1978) developed the assay further by utilizing nitroblue tetrazolium as the... 

Various colorimetric methods are also available, based on non-specific dye binding to polypeptide chains, one of the more common being the Bradford assay. One drawback with such methods is that the actual colour intensity (absorbance) developed is not absolute, but depends on the specific protein. Calibration can therefore be a problem if accurate concentrations are required. 

Fritz E, Thiele D, Willems H, Wittenbrink M-M. Quantitation of Coxiella burnetii by polymerase chain reaction and a colorimetric microtiter plate hybridization assay. Eur J Epidemiol. 1995 11 549-557. 

Vlieger AM, Medenblik AM, van Gijlswijk RP, Tanke HJ, van der Ploeg M, Gratama JW, and Raap AK (1992) Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection. Analytical Biochemistry 205(1) 1-7. 

Guglielmo-Viret, V, ThulHer, R, 2007. Comparison of an electrochemilumines-cence assay in plate format over a colorimetric ELISA, for the detection of ricin B chain. J. Immunol. Methods 328,70-78. 

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