Assays of alkaline phosphatases

The concentration of Pi in the assay mixture should be lower than 0.05 mM. Tris buffers are preferred for the bacterial APase and diethanolamine (DEA) for the bovine enzyme, which gives, with excess substrate, about 15% higher activity in a 2 M DEA-HCl buffer than at 1 M. However, the background is also stronger (Walter and Schutt, 1974). 

Mix and measure 30 sec later for 2 min (stopwatch) at 405 nm. If the increase in absorbance is significantly more than 0.2/min, dilute the sample with physiological NaCl solution (150 mM). 

Jung and Kohler (1980) reported that the molar extinction coefficient (18.6 X 103 M- cm ) of p-NP depends on the assay temperature, concentration of the buffer, and the presence of proteins. Small, apparent differences reported in activities may, therefore, be due in part to a changing extinction coefficient. 

---

Bronstein, I., Voyta, J. C., Thorpe, G. H. G., Kricka, L. J., and Armstrong, G (1989) Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin. Clin Chem 35, 1441—1446. 

In addition to the classical symptoms of zinc deficiency mentioned above, the following unusual conditions have been reported liver and spleen enlargement, abnormal dark adaptation and abnormalities of taste. Several laboratory procedures for diagnosing zinc deficiency are available. Measurement of zinc levels in plasma is useful in certain cases. Levels of zinc in the red cells and hair may be used for assessment of body zinc status. More accurate and useful parameters are neutrophil zinc determination and quantitative assay of alkaline phosphatase activity in neutrophils. Determination of zinc in 24 h urine may help diagnose deficiency if sickle cell disease, chronic renal disease and liver cirrhosis are ruled out. A metabolic balance study may clearly distinguish zinc-deficient subjects. 

Figure 36.6. Snapshot of the Method creator of the Immusoft computer programme serving for the establishment of assay protocols. The figure shows on the right-hand side the various steps of the protocol developed for the assay of alkaline phosphatase (ALP), in which the functionalisation of the microchannels (coating and blocking steps) is directly integrated in the assay progress.

The standard incubation mixture for assay of alkaline phosphatase, contained in a total 200 fiL 5 mM disodium phenylphosphate, 50 mM carbonate buffer (pH 10.2), saliva, and distilled water. The reaction was started by addition of saliva and was carried out at 37°C for 30 minutes. The reaction was... 

The attachment of the ferrocene groups to small molecules has been used in two main classes of biosensor in the first, the conjugate is an enzyme substrate such that a shift in its redox potential occurs following enzymatic modification. An example is ferrocenylethyl phosphate (Figure 9) that facilitates the electrochemical assay of alkaline phosphatase, a common enzyme label for immunoassays. The redox potential of the alcohol product is lower than the phosphate ester and hence by poising the potential, the product can be selectively measured and so the alkaline phosphatase activity measured. The sensitivity of this method was further enhanced by the use of stripping voltammetry to detect the product. ... 

Colorimetric assay of alkaline phosphatase with NADP as a substrate and using en2yme cycling. 

Pure 4 -FMN was used in an amplified colorimetric assay of alkaline phosphatase. This system allows the detection of very low amounts of enzyme in the range of 4 amol (82). 

S Harbron, HJ Eggelte, BR Rabin. Amplified colorimetric assay of alkaline phosphatase using riboflavin 4 -phosphate a simple method for measuring riboflavin and riboflavin 5 -phosphate. Anal Biochem 198 47-51, 1991. 

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

Liver injury is clinically defined as an increase of serum alanine amino transferase (ALT) levels of more than three times the upper limit of normal and a total bilirubin level of more than twice the upper limit of normal [4]. The clinical patterns of liver injury can be characterized as hepatocellular (with a predominant initial elevation of ALT), cholestatic (with an initial elevation of alkaline phosphatase) or mixed. The mechanisms of drug-induced hepatotoxicity include excessive generation of reactive metabolites, mitochondrial dysfunction, oxidative stress and inhibition of bile salt efflux protein [5]. Better understandings of these mechanisms in the past decades led to the development of assays and models suitable for studying such toxic mechanisms and for selecting better leads in the drug discovery stage. 

In the application of alkaline-phosphatase-sensitive, triggerable 1,2-dioxetanes, the nucleic-acid hybridization assay is nowadays quite popular . Such techniques include viral load assays for hepatitis B and C and for human immunodeficiency viruses (HBV,... 

Alkaline phosphomonoesterase (EC 3.1.3.1). The existence of a phosphatase in milk was first recognized in 1925. Subsequently characterized as an alkaline phosphatase, it became significant when it was shown that the time-temperature combinations required for the thermal inactivation of alkaline phosphatase were slightly more severe than those required to destroy Mycobacterium tuberculosis, then the target micro-organism for pasteurization. The enzyme is readily assayed, and a test procedure based on alkaline phosphatase inactivation was developed for routine quality control of milk pasteurization. Several major modifications of the test have been developed. The usual substrates are phenyl phosphate, p-nitrophenyl-phosphate or phenolphthalein phosphate which are hydrolysed to inorganic phosphate and phenol, p-nitrophenol or phenolphthalein, respectively ... 

CIEEL is of particular interest for the development of modern chemiluminescent bioassays. The most popular clinical bioassays utilize thermally persistent spiro-adamantyl-substituted dioxetanes with a protected phenolate moiety. These designed 1,2-dioxetanes include an energy source, a fluorophore, and a trigger grouping, and are therefore structurally similar to bioluminescent substrates such as firefly luciferin. Three main commercial dioxetanes 75 are available as one-reagent assays for alkaline phosphatase and are sold under the name of AMPPD (R1 = R2 = H), CSPD (R1 = Cl, R2 = H), and CDP-Star (R1 = R2 = Cl) <2006S1781, 2003ANA279>. These substrates are sensitive to 10 21 mol of alkaline phosphatase in solution. 

A single-ceU fluorescent assay for expression of alkaline phosphatase (Dyhrman and Palenik, 1999), has been used to show that Trichodesmium populations in the Atlantic are likely phosphate-deficient and largely growing on DOP sources (Dyhrman et al., 2002). These results are supported by several other studies showing that Trichodesmium is capable ofefEciendy using DOP (Stihl etal, 2001 Yentsch etai, 1972). MulhoUand... 

Substrate to give a soluble product (plate assays) 0.1% / -Nitrophenyl phosphate in 10 mAfdiethanolamine, pH 9.5, containing 0.5 mAfMgClg. Substrate to give an insoluble product (Western blots) NBT stock— 5% nitroblue tetrazolium in 70% dimethyl formamide. BCIP stock—5% disodium bromochloroindolyl phosphate in dimethyl formamide. Alkaline phosphatase buffer—100 mAf diethanolamine, pH 9.5, containing 100 mAfNaCl and 5 m AfMgClj. Just before use, add 66 lL of NBT stock solution to 10 mL of alkaline phosphatase buffer, mix well, and add 33 pL of BCIP stock soludon. 

Enzyme DNA hybridization assays with electrochemical detection can offer enhanced sensitivity and reduced instrumentation costs in comparison with their optical counterparts. Efforts to prevent non-specific binding of the codissolved enzyme and to avoid fouling problems by selecting conditions suitable to amplify the electrode response have been reported by Heller and co-workers [107]. A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode was described by Limoges and co-workers for an enzyme nucleic acid hybridization assay using alkaline phosphatase [108] or horseradish peroxidase [109]. In another methodology to improve sensitivity, a carbon paste electrode with an immobilized nucleotide on the electrode surface and methylene blue as hybridization indicator was coupled, by Mascini and co-workers [110], with PGR amplification of DNA extracted from human blood for the electrochemical detection of virus. 

EI2I Norton, G.E., Thunberg, A.L., Dappen, G.M., LaRossa, D.D., Snoke, R.E. and Schubert, R.M. (1983). Development of Kodak Ektachem clinical chemistry slide assay for alkaline phosphatase. Clin. Chem. 29, 1269-1270, Abstr. 259. 

ST53 Butch, A.W., Goodnow, T.T., Brown, W.S., McCellan, A., Kessler, G. and Scott, M.G. (1989). Stratus automated creatine kinase-MB assay evaluated Identification and elimination of falsely increased results associated with a high-molecular-mass form of alkaline phosphatase. Clin. Chem. 35, 2048-2053. 

Garnero P, Delmas PD. Assessment of the serum levels of alkaline phosphatase witli a new immunora-. diometric assay in patients with metabolic bone disease. J Clin Endocrinol Metab 1993 77 1046-53. 

A heterogeneous solid-phase enzyme immunoassay for DHEA-S is commercially available. This method uses horseradish peroxidase as enzyme label, rabbit anti-DHEA-S-coated microtiter wells, and tetramethylbenzidine as substrate. The assay is stated to have a lower limit of detection of 15ng/dL (40.6nmol/L). An automated method employing chemiluminescent detection of alkaline phosphatase-labeled conjugate has been described. This assay reported a lower limit of detection of 2pg/mL (54.0nmol/L). 

Several assays, such as the ultra-sensitive luminescent ELRA developed by Seifert (2004) with a detection limit of 20 ng L-1 for 17 (3-estradiol, have been reported in the literature. Although these assays are simple to use, many factors, such as differences in culture conditions, cell density or cell-line clones, affect the potency of estrogenic substances, and that makes the standardization of these methods difficult. The induction of several proteins or enzyme activities (e.g. the increasing levels of alkaline phosphatase, cathepsin D, prolactin and vitellogenin as a consequence of progestogens) has also been used to study estrogenicity. However, expression of these proteins or enzyme activities is restricted to specific cell lines and cannot be extrapolated to other tissues or species. 

---