Time-Resolved Fluorescence-Based Assay for the Determination of Alkaline Phosphatase Activity and Application to the Screening of Its Inhibitors

A single-step end point method is presented for determination of the activity of the enzyme alkaline phosphatase (ALP) using the effect of enhancement of fluorescence of the easily accessible europium(III)-tetracycline 3:1 complex (Eu3TC). Its luminescence, peaking at 616 nm if excited at 405 nm, is enhanced by a factor of 2.5 in the presence of phosphate. Phenyl phosphate was used as a substrate that is enzymatically hydrolyzed to form phenol and phosphate. The latter coordinates to Eu3TC and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. The assay is performed in a time-resolved (gated) mode, which is shown to yield larger signal changes than steady-state measurement of fluorescence. The limit of detection for ALP is 4 µmol L—1. Based on this scheme, a model assay for theophylline as inhibitor for ALP was developed with a linear range from 14 to 68 µmol L— 1 of theophylline. ( Journal of Biomolecular Screening 2008:9-16)

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Determination of Anti-Acetylcholine Receptor Antibodies in Myasthenic Patients by Use of Time-resolved Fluorescence

Clinical Chemistry ◽

10.1093/clinchem/48.3.549 ◽

2002 ◽

Vol 48 (3) ◽

pp. 549-554 ◽

Cited By ~ 7

Author(s):

Jan Říčný ◽

Libuše Šimková ◽

Angela Vincent

Keyword(s):

Acetylcholine Receptor ◽

Acetylcholine Receptors ◽

Limit Of Detection ◽

Linear Regression Analysis ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

Use Of Time ◽

A Value ◽

Restricted Use

Abstract Background: Autoantibodies against nicotinic acetylcholine receptor (nAChR) in myasthenia gravis (MG) patients are usually detected by radioimmunoprecipitation assays using extracted acetylcholine receptors labeled irreversibly with 125I-α-bungarotoxin (α-BuTx). To provide a nonradioactive immunoassay, we established an assay using nAChRs labeled with Eu3+-α-cobratoxin (α-CTx). Methods: We derivatized α-CTx with a diethylenetriaminepentaacetate moiety and formed a complex with Eu3+. The complex was purified by HPLC, and the fractions were tested for binding to Torpedo and human nAChRs. The most active fractions were used to label nAChRs for the immunoprecipitation assay, and the bound Eu3+ was quantified by time-resolved fluorescence. Results: Eu3+-labeled α-CTx competed with 125I-α-BuTx for binding to Torpedo nAChRs and saturated the binding sites of human nAChRs, with a Kd of 7.2 × 10−9 mol/L. Results of the immunoassay performed with Eu3+-labeled α-CTx were similar to those obtained with 125I-α-BuTx, with a slightly higher limit of detection [0.3 nmol/L (n = 6) vs ∼0.1 nmol/L for isotopic assay]. None of 34 negative sera tested (16 healthy controls, 10 patients with nonmyasthenia-related disease, 8 patients seronegative for MG) gave a value >0.3 nmol/L. Of the 35 positive myasthenic sera (with antibody values, previously determined by isotopic assay, of 0.4–1290 nmol/L) compared in the two assays, 32 tested positive with the Eu3+ assay. Linear regression analysis yielded the equation: y = 1.035x − 0.013 nmol/L; Sy|x = 0.172 nmol/L; r2 = 0.977. Conclusions: The new time-resolved fluorescence method for quantification of antibodies to nAChRs in MG patients provides a performance similar to that of the widely used isotopic assay and could be used in laboratories with restricted use of isotopes.

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