Assay for alkaline phosphatase

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. 

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. 

Fig. 2 Brush border enzyme activity in primary rat proximal tubular cells (RPTC) as a function of age. RPTC were cultured in 35 mm culture dishes for 4,6,8,10, and 12 days. Cell homogenates were assayed for alkaline phosphatase activity (ALP) and gamma glutamyl transpeptidase activity (GGT). Values were correlated to homogenate protein content and are expressed as a fraction of isolated proximal tubular fragment activity/mg protein (day 0). Results are the mean of three culture dishes from two different primary preparations. Statistical significance from day 0 as analyzed by one way ANOVA with a Dunnett s post test is denoted by where p<0.05 and by where p<0.01...

Hfll HD, Summer GK, Waters MD (1968) An automated fluorometric assay for alkaline phosphatase using 3-O-methylfluorescein phosphate. Anal Biochem 24 9—17... 

The cytochemical assay for alkaline phosphatase activity is semiquanti-tative, and the absolute results were not expected to correspond to the direct measurement of enzyme activity in supernatants surrounding sheared... 

Immunoassays presently form probably the most active area of exploitation of CL, ECL and BL, as evidenced by the extensive commerciahzation of luminescent immunoassay analyzers and test kits. Among these, acridinium ester-based CL assays for alkaline phosphatase and amplified CL assays using peroxidase labels have become the most widespread [207, 208]. Besides immunosassays CL and BL have been used in nucleic acid assays [209-211] and in cellular studies concerning, for instance, phagocytosis [212]. ECL immunoassays based on ruthenium trisbipyridyl labels are now becoming popular in clinical immunoassay analyzers [213]. 

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. 

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). 

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. 

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. 

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. 

The most common enzyme label in IAs and ILAs is horseradish peroxidase (HRP) due to its high turnover number, the sensitivity of its colorimetric and luminometric assays, its suitability for diverse conjugation procedures, and relative small molecular size (40 kDa compared to 100 kDa for alkaline phosphatase). The use of labeled enzymes as tracers allows qualitative and quantitative assay procedures that are not dependent on instrumentation. Thus absorbance, luminescent, electrochemical, or multistage assay systems could be performed (Fig. 10) [23]. 

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.

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). 

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... 

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. 

Chemiluminescence assays are ultrasensitive (attomole to zeptomole detection limits) and have wide dynamic ranges. They are now widely used in automated immunoassay and DNA probe assay systems, (e.g., acridinium ester and acri-dinium sulfonamide labels and 1,2-dioxetane substrates for alkaline phosphatase labels and the enhanced-luminol reaction for horseradish peroxidase labels [see Chapter 9]). 

To solve the problem, we have developed a highly specific and sensitive assay for C-peptide in serum and plasma using specific monoclonal antibody (MoAb) to the N-terminal of the C-peptide molecule. In this report, we describe the assay performance of C-peptide on the LUMIPULSE system. The system is a fully automated chemiluminescent enzyme immunoassay (CLEIA) system that uses AMPPD as a substrate for alkaline phosphatase and ferrite micro-particles as a solid phase. ... 

Olesen CEM. Dioxetane substrate for alkaline phosphatase labels. J Clin Ligand Assay 22, 1999 2 129-38. 

Amino-alcohol buffers are used commonly for alkaline phosphatase (ALP, EC 3.1.3.1) assays. Some sources of these buffers are contaminated with a potent ALP inhibitor, an obvious interferant (46). 

Infectious Diseases DNA hybridization assays that employ dioxetane substrates for alkaline phosphatase were first briefly reported in 1988, viz., herpes simplex 1 vims (B21) and Chlamydia trachomatis (Cl 7). These two assays were followed by an assay for hepatitis B vims core antigen, HBVc, (B18, B26). Thus, 10 copies of HBVc-containing plasmid DNA were detected in 30 min in a dot-blot assay, whereas a colorimetric test required 10 copies in the same time (B18, B26). Optimization allowed 10 -10 copies to be detected in 2 hr. An in situ hybridization assay for HSV-1 detected its presence after only 5 min (B22). A more detailed account of the Chlamydia assay describes how probes were prepared against two sites on an endogenous 7.4-kb plasmid and how, in a 5-hr assay. 

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. ... 

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