Naphthol phosphate

Develop the chromogeuic reaction of the second sequence using AEC/H2O2 development for IPO or naphthol phosphate/Fast Blue, Fast Red, or New Fuchsin for lAP development as detailed in Subheading 3.3. (see Note 15 for triple labeling). 

In the immunoalkaline phosphatase staining method, the enzyme hydrolyzes naphthol phosphate esters (substrate) to phenolic compounds and phosphates. 

Fast Red-naphthol phosphate standardized chromogen-substrate kit (e.g., Dako, code K0597). 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

CL reaction can be catalyzed by enzymes other than HRP (e.g., microperoxidase and catalase) and by other substances [hemoglobin, cytochrome c, Fe(III), and other metal complexes]. The presence of suitable molecules such as phenols (p-iodophenol), naphthols (l-bromo-2-naphthol), or amines (p-anisidine) increases the light production deriving from the HRP-catalyzed oxidation of luminol and produces glow-type kinetics [6, 7], The use of other enzymes, such as glucose-6-phosphate dehydrogenase [38-41], P-galactosidase [42], and xanthine oxidase [43-46], as CL labels has been reported. 

Developer Naphthol AS-BI phosphate (Sigma N4875) 2 mg, N,N-dimeth-ylformamid 0.2 mL, veronal-acetate buffer 9.8 mL, pH 9.2, levamisol 1 M... 

Develop with (naphthol AS-BI phosphate/Fast Red, fresh made) for 20 min in the dark (in tinfoil-sealed humidity chambers). For preparation of developer, see Subheading 5.1.1.1, item 4. 

Dibasic calcium phosphate 0.2 g Hydroxy naphthol blue Each ml of 0.05 M disodium edetate = 0.002004 g of Ca... 

Chemical/Physical. Decomposes at elevated temperatures forming methyl isocyanate (Windholz et al., 1983) and nitrogen oxides (Lewis, 1990). Hydrolyzes in water to 1-naphthol and 2-iso-propoxyphenol (Miles et al., 1988). At pH 6.9, half-lives of 78 and 124 d were reported under aerobic and anaerobic conditions, respectively (Kanazawa, 1987). Miles et al. (1988) studied the rate of hydrolysis of propoxur in phosphate-buffered water (0.01 M) at 26 °C with and without a chlorinating agent (10 mg/L hypochlorite solution). The hydrolysis half-lives at pH 7 and 8 with and without chlorine were 3.5 and 10.3 d and 0.05 and 1.2 d, respectively. The reported hydrolysis half-lives of propoxur in water at pH 8, 9, and 10 were 16.0 d, 1.6 d, and 4.2 h, respectively (Aly and ELDib, 1971). In a 0.50 N sodium hydroxide solution at 20 °C, the hydrolysis half-life was reported to be 3.0 d (ELDib and Aly, 1976). 

Alkaline phosphatase acts on many substrates as well, each precipitating as a different color. For example, a combination of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) results in a permanent blue precipitate at the site of alkaline phosphatase localization. There are other compounds that can also be tried, such as Fast Red TR/Naphthol AS-MX (Sigma, St. Louis, MO), which precipitates as a red color. 

A disposable electrochemical enzyme-amplified genosensor was described for specific detection of Salmonella (Del Giallo et al., 2005). A DNA probe specific for Salmonella was immobilized onto screen-printed carbon electrodes and allowed to hybridize with a biotinylated PCR-amplified product of Salmonella. The hybridization reaction was detected using streptavidin conjugated-AP where the enzyme catalyzed the conversion of electroinactive a-naphthyl phosphate to electroactive a-naphthol, which was detected by differential pulse voltammetry. 

An interesting variation of the latter technique finds application in enzyme chemistry. In this procedure a tissue section is exposed to a relatively colorless derivative of j8-naphthol, such as sodium j8-naphthyl acid phosphate. A phos-photase enzyme reacts with this reagent (often called an enzyme substrate ), leaving free j8-naphthol behind. Subsequent treatment with a solution of a diazonium salt produces highly colored spots in the tissue section. Thus not only can the presence of phosphotase enzyme be demonstrated, but also the location of the enzyme in the tissue can be determined. The intensity and chroma of the color produced and the solubility of the azo dye in the cell materials can be varied by judicious selection of the reagents. 

Develop the alkaline phosphatase with fast red dissolve 5 mg sodium naphthol AS BI phosphate in dimethylformamide (few drops) and add to 5 mg fast red TR salt in 10 ml veronal acetate buffer (pH = 9.2), incubate the slides for 1 h Wash with tap water Counterstain as desired... 

A section of the dorsal part is shown (p = parenchyma t = tegument). Immunolocalization was performed with an antibody against Schistosoma mansoni Hsp70 (Moser ef a/., 1990). Detection was performed using an alkaline-phosphatase conjugated secondary antibody, naphthol-AS-phosphate and Fast Red TR (Finken ef a/., 1994). 

Most of the older methods of fluorimetric analysis of pesticides involved hydrolysis to form fluorescent anions. Co-ral (coumaphos) [147] was hydrolyzed in alkali to the hydroxybenzopyran, which was subsequently determined by means of its fluorescence. Guthion (azinphosmethyl) was hydrolyzed to anthranilic acid for fluorimetric analysis [148,149]. A method was developed [150] for Maretin (N-hydroxynaphthalimide diethyl phosphate) in fat and meat which involved hydrolysis in 0.5 M methanolic sodium hydroxide followed by determination of the fluorescence of the liberated naphthalimide moiety. Carbaryl (1-naphthyl N-methylcarbamate) and its metabolites have been determined by a number of workers using base hydrolysis and the fluorescence of the resulting naphtholate anion [151-153]. Nanogram quantities of the naphtholate anion could be detected. Zectran (4-dimethylamino-3,5-xylyl N-methylcarbamate) has been determined by the fluorescence of its hydrolysis product [154]. The fluorescence behaviour of other carbamate insecticides in neutral and basic media has been reported [155]. Gibberellin spray used on cherries has been determined fluorimetrically after treatment with strong acid [156]. Benomyl (methyl N-[l-(butylcarbamoyl)-2-benzimidazolyl]carbamate) has been analyzed by fluorimetry after hydrolysis to 2-aminobenzimidazole [157]. 

Note PAN = 1-(2-pyridylazo)-2-naphthol DMG = dimethylglyoxime DPC = 1,5-diphenylcarbazide DADTC = diethylammonium diethyldithiocarbamate PMBP = 1-phenyl-3-methyl-4-benzoyl-pyrazolone-5 HDEHP = b/s-[2-ethylhexyl]phosphate TBP = tributyl phosphate. 

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