Phosphatases assay methods

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... 

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). 

Urinary alkaline phosphatase activity. II. An analytical validation of the assay method. JAMA (1963), 185, 953-957. 

HCV and HIV-1). The bDNA assay is being much employed for the quantification of messenger RNA. Moreover, for the detection of viral and pathogenic disorders based on alkahne-phosphatase-sensitive dioxetanes, several assay methods are available these include the Polymerase-Chain-Reaction (PCR) amphfication, probe ligation, strand-displacement amplification and the ligase chain reaction. ... 

Montalibet, J., K.I. Skorey, and B.P. Kennedy. 2005. Protein tyrosine phosphatase enzymatic assays. Methods 35, 2-8. 

The mouse bioassay, which is still the ofircial method used for some natural toxins present in the marine enviromnent, has been used as a routine screening method for a few years, but this method has been recently replaced in maty laboratories worldwide with a more sensitive method such as an enzyme-linked-immrmosorbent assay (Nagata 1995). Protein phosphatase assays (Metcalf 2001) have been also used but more details on these methods will be discussed later. 

The results obtained with a protein phosphatase assay and a HPLC/UV/MS method are compared with the results obtained with a bioluminescence assay, which is successfully introduced here for... 

A control solution of 8xl0 M alkaline phosphatase was prepared. A second solution of 8xl0 ° M enzyme was heated at 64°C for 5 min and immediately diluted by 6 orders of magnitude. Both samples were assayed by the single molecule assay method. 

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. 

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. 

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). 

To solve the problems arising from the use of protein phosphatases and ELISAs, a new colorimetric immunoprotein phosphatase assay (CIPPIA) for specific detection of microcystins and nodu-larins of cyanobacteria was developed by Metcalf et al. [170]. The principle of this method is based... 

Detection methods for the enzyme reactions include colorimetric, fluorometric, chemiluminometric, and bioluminometric assays, as well as electrochemical methods, and their detection ranges vary. The principal enzymes used for labeling, assay methods available, and levels of sensitivity are summarized in Table 1. Amongst these, horseradish peroxidase, j8-D-galactosidase from Escherichia coli (E. coli), and alkaline phosphatase from calf intestine or E. coli are currently the most widely used for conjugating antigens or antibodies. 

Another common procedure for stopped assays is the use of molybdate under acid conditions to complex and spectrophotometrically assay inorganic phosphate. This provides a usefiil alternative to jMiitrophenyl phosphate for phosphatase assays, as physiologically relevant phosphate esters can be used. For example, this assay also provides a useful method for measuring ATPase activity. Details of the phosphomolybdate assay arc given here (however, see Section 8.5 for methodologies that utilize plate readers). 

The acid phosphatase (ACP) method, developed by Bellavite et al. [151], is based on acid phosphatase platelet activity. Acid phosphatase is a stable enzyme present in platelets. This method is similar to the LDH method. It uses regular PRP and the same Triton-X to completely lyse attached platelets. But in this test, a different enzyme (acid phosphatase) is measnred. On addition of p-nitrophenyl phosphate snbstrate, acid phosphatase converts the snbstrate into p-nitrophenol, which can be easily measnred by a photospectrometer at a wavelength of 405 nm. Side-by-side comparison of both DCH and ACP assays snggests that the ACP assay is better than the LDH assay in terms of reproducibility [152]. 

Xsukatani, X. Ide, S. Ono, M. Matsumoto, K. New tetrazolium method for phosphatase assay using ascorbic acid 2-phosphate as a substrate. Talanta... 

For most assays, the incorporated pantothenic acid has to be Hberated en2ymatically. Usually, a combination of pantotheinase and alkaline phosphatase is used to hberate the bound pantothenic acid. The official method for pantothenic acid of the Association of Official Analytical Chemists (AOAC) is the microbiological assay that uses U. Plantarium (A.TCC 8014) as the test organism (71). Samples are extracted at 121°C at pH 5.6—5.7, proteins are precipitated at pH 4.5, and the resulting clear extracts are adjusted to pH 6.8 prior to assay. This procedure is only suitable to determine calcium pantothenate or other free forms of pantothenic acid. 

After screening for toxicity, identification and/or quantification assays may need to be carried out if the screening method is not specific for the cyanobacterial toxin(s) under investigation. Suitable assays for these purposes include the physicochemical assays, HPLC, MS, and CE, and to some extent the immunoassays and protein phosphatase inhibition assays summarized in Section 2. 

---