Alkaline phosphatase assay phosphate

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. 

Dyhrnian, S. T., and Palenik, B. (1999). Phosphate stress in cultures and field populations of the dinoflageUate Prorocentrum minimum detected by a single-ceU alkaline phosphatase assay. Appl. Environ. Microbiol. 65, 3205—3212. 

Related to this, the Abe group reported earlier on the interaction between PEC precipitate systems consisting of anionic phosphated and carboxymethylated chitin (PCHN and CCHN) and cationic CHT and rat OB cells in cell culture dishes [174]. OB aggregates were observed on both CHT/PCHN (4th day) and CHT/CCHN (2nd day) to exhibit inhibited growth as compared with controls. However, OB differentiation (as shown by alkaline phosphatase assay) increased and osteocalcin mRNA was expressed for both PEC systems, indicating the importance of OB aggregation for bone mineralization capabihty. 

Recently a number of phosphate accepting buffers have been used in alkaline phosphatase assays, onto which the liberated phosphate group is attached. These are amino alcohols such as diethanolamine, tris and ethylaminoethanol. When present they can enhance the rate of reaction. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Cambella and Antia [385] determined phosphonates in seawater by fractionation of the total phosphorus. The seawater sample was divided into two aliquots. The first was analysed for total phosphorus by the nitrate oxidation method capable of breaking down phosphonates, phosphate esters, nucleotides, and polyphosphates. The second aliquot was added to a suspension of bacterial (Escherichia coli) alkaline phosphatase enzyme, incubated for 2h at 37 °C and subjected to hot acid hydrolysis for 1 h. The resultant hot acid-enzyme sample was assayed for molybdate reactive phosphate which was estimated as the sum of enzyme hydrolysable phosphate and acid hydrolysable... 

The Abbott IMx , a dedicated commercial immunoassay analyzer that employs FPIAs for small molecules, can also determine larger analytes by a fluorescence-based microparticle capture enzyme immunoassay (MEIA).(44) In this system, antibody-coated0.47- mlatexparticles are used for both sandwich and competitive assays, and alkaline phosphatase conjugates that bind to the particles cleave 4-methylumbelliferyl phosphate to generate the fluorophore. 

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

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 ... 

Fig. 5. Activation of calf intestinal alkaline phosphatase by Mg. Assays were performed at 38° in 0.05 M ethanolamine-HCl pH 9.9 with 2.5 mM phenyl phosphate. From Morton (90).

Protection of the phenolic moiety as a phosphate provides substrates such as 58 that are specifically recognized and cleaved by alkaline phosphatase to afford 59 (Scheme 13) <1998PCA5406>. This type of dioxetane is behind many of the commercial assays that are discussed further in Section 2.16.10.1. 

A low detection limit directly influences the sensitivity of the enzyme-based assay. The final enzyme-substrate interaction must yield an ample amount of some end product which can be accurately monitored and, hopefully, quantitated. The authors experiences have been chiefly with enzymatic detection systems which culminate in a visible chromogenic reaction (e.g. alkaline phosphatase, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate). 

Luminescent endpoints are available for all of the commonly used enzyme labels, including horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, glucose oxidase, and P-galactosidase. Detection limits in the subattomolar range have been attained (34). Chemiluminescent assays for novel enzyme labels such as xanthine oxidase have also been developed (50). 

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... 

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