Alkaline phosphatase assay

RIPA lysis buffer (without vanadate and sodium fluoride inhibitor as they inhibit phosphatase activity) 

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Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

Data obtained from 16 replicate alkaline phosphatase assays that were measured within a single rotor Results are given in I.U., 30° C. 

Alkaline phosphatase assays based on 3-glycerophosphate now appears to be obsolete, and methods buffered by either glycine or barbital are also obsolete as these buffers inhibit ALP or are poor buffers. Serum alkaline phosphatase is known to be composed of several isoenzymes which presumably arise from bone, liver, intestine, and placenta. The placental alkaline phosphatase is known to be much more resistant to heat denaturation than the other isoenzymes, and this resistance provides a simple test for it (5). The other enzymes can be separated through the differential inhibition by phenylalanine, by electrophoresis and by specific antibodies. However, the clinical usefulness of the results obtained is in doubt (23). 

Figure 36.7. Example of Immusoft window appearing in the Analysis menu during the processing of the fluidic steps of an assay. The figure shows the electrochemical monitoring of the fluid flux within the microchannels obtained during the 30 loading steps of the microchannel coating procedure chosen for alkaline phosphatase assays.

A solution of substrate (50 pL/well) is added and the plates incubated in the absence of light (time varies from case to case, but is usually 15-30 min). After incubation, the reacted solution is collected in an ELISA plate by centrifuging at 335g for 1 min, and the absorbance of the samples read at the characteristic wavelength (e.g., 405 nm for alkaline phosphatase assays). 

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. 

Standard alkaline phosphatase assay has shown that heating of the enzyme for 5 min at 64°C results in a 50% loss of activity. This loss in activity may result from 2 possibilities Total denaturation of half the molecules or partial denaturation of all the molecules. 

A cascade amplification method has been proposed for improving the detection limit of the alkaline phosphatase assay, and involves a different synthetic substrate (Eq. 3.21), (oxotrifluorobutyl)phenylphosphate 11... 

The substrate for the indicator reaction, dichloroindophenylbutyrate, is pale yellow. The product of the reaction is deep blue, with an absorbance maximum at 620 nm. The ingenuity of this method lies in the production of an enzyme inhibitor, (oxotrifluor-obutyl)phenol, rather than a species that is directly quantitated. The inhibitor affects the activity of the esterase, resulting in catalytic amplification of the initial alkaline phosphatase activity. The detection limit of the amplified assay has been reported as 3.2 x 10 14M, a 100-fold improvement over the detection limit of the standard alkaline phosphatase assay. 

Bouman AA, Scheffer PG, Ooms ME, Lips P, Netelenbos C. Two bone alkaline phosphatase assays compared with osteocalcin as a marker of bone formation in healtliy elderly individuals. Clin Chem 1995 41 196-9. 

The above observations call for the adoption of a serum alkaline phosphatase assay method in which either an undiluted human serum is used or a constant dilution of sera is maintained. This is necessary to obtain comparable and reproducible results with different serum specimens. Fishman (F5, F6) avoided this difiiculty by using a solution of... 

Starch-gel electrophoresis of the alkaline phosphatase in the butanol extracts of leukocytes revealed three variants of the enzyme. Peacock et al. (PI) have devised a method for leukocyte alkaline phosphatase assay. An additional variant was detected in blood leukocytes of leukemia patients treated with 6-mercaptopurine (RIO). Robinson and Pierce (R7) indicated that there might be a fundamental difference in molecular structure of the human serum alkaline phosphatase proteins because serum alkaline phosphatase, when incubated with neuraminidase prior to electrophoresis, demonstrated reduced anodal migration of those isoenzymes that are not L-phenylalanine-sensitive. L-Phenylalanine-sensi-tive enzyme of intestinal origin was found to be neuraminidase-resistant. 

We performed a series of experiments to elucidate the MOA of the novel TNAP inhibitors. The catalytic mechanism by which TNAP degrades PR consists of rapid phosphorylation of the active site in the presence of the phospho-donor substrate and a rate-limiting dephosphorylation by the phospho-acceptor substrate, either water or amino-containing alcohols (Fig. 5). All the TNAP inhibitors reported to date are uncompetitive with respect to phos-pho-donors and are likely to be non- or uncompetitive with diethanolamine (DBA). The latter conclusion is based on the fact that the majority of the alkaline phosphatase assays are performed in the presence of saturating concentrations of DBA or other phosphor-acceptors. 

Eade, M. N., Cooke, W. T., and Williams, ]. A., Liver disease in Crohn s disease, A study of 100 consecutive patients. Scand.. Gastroenterol. 6, 199-204 (1971). Eaton, R. H., Plasma alkaline phosphatase assay. Interconversion of results by two methods. Clin. Chem. 23, 2148-2150 (1977). 

Figure 2. Luminescent curve of new 1,2-dioxetane derivative and AMPPD (During 5 min incubation with alkaline phosphatase assay)...

The activity of compound 41 was reversed to agonism by changing the carbonyl group into a thiocarbonyl group (42). Compound 42 was agonistic in vitro with a potency in the alkaline phosphatase assay of 0.7 nM. 

Durrieu, C., Badreddine, I. and Daix, C. (2003) A dialysis system with phytoplankton for monitoring chemical pollution in freshwater ecosystems by alkaline phosphatase assay. Journal of Applied Phycology 1 5, 289-295. 

The fluorescent assay for alkaline phosphatase takes advantage of a related reagent, 4-methyl-umbelliferylphosphate. As in the case of /1-D-galacto-sidase, enzyme activity produces the fluorescent product 4-methylumbelliferone. However, 4-methyT umbelliferylphosphate has a relatively high intrinsic fluorescence. As a result, the fluorometric alkaline phosphatase assay is not as sensitive as the fS-u-galactosidase assay. 

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. 

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