Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system

Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.     

2-Carboxy-1-naphthyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase

A new substrate, 2-carboxy-1-naphthyl phosphate (CNP), was developed for the fluorimetric determination of alkaline phosphatase (ALP) activity. The product of the enzyme reaction is 1-hydroxy-2-naphthoic acid (HNA), which is a strong fluorescent product. The amount of HNA generated is proportional to ALP activity. Optimal conditions for the determination of ALP were investigated. The linear range and detection limit for the determination of ALP are 0.01-4.8 U/L and 7.44 mU/L, respectively. This method is simple, practical and can be successfully applied to assess ALP in human serum with good accuracy and precision. The results were evaluated by comparison with a standard colorimetric assay using p-nitrophenyl phosphate as ALP substrate.     

Spectrophotometric enzyme-amplified immunoassay for thyroid stimulating hormone.

Thyroid stimulating hormone (TSH) regulates the function of the thyroid gland. Its determination at low concentrations in serum is useful in the diagnosis of hyperthyroidism. In this paper, it is detected using a spectrophotometric enzyme-amplified immunoassay. The reporter enzyme is alkaline phosphatase and its substrate is flavin adenine dinucleotide phosphate (FADP). Reaction with alkaline phosphatase converts FADP into flavin adenine dinucleotide (FAD), which, unlike FADP, re-activates apo-D-amino acid oxidase (apo-AOD). Re-activation of apo-AOD allows the product of the reporter enzyme to be amplified. The lower limit of detection for TSH by this method is 0.06 microU cm-3. This compares with 0.54 microU cm-3 for an identical assay in which p-nitrophenyl phosphate was the substrate for alkaline phosphatase. Contaminating alkaline phosphatase was removed from the reagents by affinity chromatography.     

Analysis of calcineurin activity by capillary electrophoresis with laser-induced fluorescence detection using peptide substrate

Reversible protein phosphorylation and dephosphorylation are very important activities in understanding cellular signaling networks. In this paper we described a CE-LIF-based assay method of calcineurin (CN), a protein phosphatase important in cardiac hypertrophy, in which a fluorescence-labeled 19-amino acid phosphopeptide was used as a substrate. The substrate was converted to a dephosphorylated product by CN and both the substrate and product were detected by the LIF detector. This assay method was tested for various separation parameters as well as reaction parameters. It was found that 100 mM of a boric acid buffer with a pH of 9.00 produced optimum separation at 10 kV of applied voltage using a 47 cm capillary. After obtaining the suitable reaction conditions the method was used to detect and quantify the CN activity in HL-1 cell extracts where the picogram level of CN activity was obtained per microgram total protein. It was also observed that immunosuppressive drugs like okadaic acid and cyclosporine A inhibit in vitro CN activity.     

Electrochemical immunoassay: an ultrasensitive method.

Hydrodynamic electrochemical techniques such as liquid chromatography and flow injection analysis with electrochemical detection are very effective for the rapid determination of the enzyme-generated product in enzyme immunoassays. The authors have used this detection method in various assay formats using both alkaline phosphatase and glucose-6-phosphate dehydrogenase as labels. Assays for digoxin will be used illustratively. Recently, the authors have used 70 mL microcapillary hematocrit tubes as the immunoassay reaction vessel and alkaline phosphatase as the labeling enzyme. The assay, complete in 30 min, had a detection limit of 5,6 x 10 -20 moles of IgG in serum. The linear range was four orders of magnitude. This low detection limit is due to a combination of the favorable geometry of the reaction vessel and the suppression of nonspecific adsorption by the addition of ion-pairing blocking agents. Even lower detectable amounts should be achievable with smaller reaction vessels. The capability for detecting such small amounts of analyte is potentially useful for the analysis of extremely small samples such as single cells and blood samples from premature infants.     

New tetrazolium method for phosphatase assay using ascorbic acid 2-phosphate as a substrate

A new method to assay alkaline and acid phosphatases assay using ascorbic acid 2-phosphate (AsA-P) and ditetrazolium salt nitroblue tetrazolium chloride (NBT) was developed. AsA-P is hydrolyzed in the presence of phosphatase to yield ascorbic acid. In turn, the ascorbic acid reduces NBT directly or indirectly, opening the tetrazole ring to produce an insoluble formazan as a colored precipitate. The proposed method for alkaline phosphatase was compared with a conventional method in which 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used in combination with NBT in the dot blots of a dilution series of β-lactoglobulin. AsA-P reduced NBT more effectively than BCIP in the presence of alkaline phosphatase. AsA-P could be also used as the chromogenic substrate for an acid phosphatase assay in the presence of phenazinium methylsulfate and NBT.     

Analysis of angiotension-converting enzyme by capillary electrophoresis.

A method is described for determination of serum angiotension-converting enzyme by capillary electrophoresis (CE) based on incubation of the substrate, a synthetic peptide, with the serum outside the capillary and cleaving hippuric acid and a dipeptide. The reaction is stopped by the addition of acetonitrile, followed by injection of the supernatant on the capillary. The acetonitrile allows injection of a large volume of sample on the capillary. Both the substrate and the reaction product (hippuric acid) can be monitored at the same time. The CE step is rapid and can be performed in about 6 min. The CE method compared well to a kinetic assay method (= 0.98).     

Capillary electrophoretic analysis of alkaline phosphatase inhibition by theophylline

An analytical method for studying enzyme inhibition has been developed using capillary electrophoresis with laser-induced fluorescence detection. This technique is based on electrophoretic mixing of zones of enzyme and inhibitor in substrate-filled capillaries. Enzyme catalytic activity is measured by detecting the fluorescent reaction product as it migrates past the detector. Reversible enzyme inhibition is indicated by a transient decrease in product formation. The enzyme, alkaline phosphatase, has been studied using the fluorogenic substrate AttoPhos ([2,2'-bibenzothiazol]-6-hydroxy-benzthiazole phosphate). This assay has been used to quantify theophylline, a noncompetitive, reversible inhibitor of alkaline phosphatase. The detection limit for theophylline is estimated at 3 microM, and 8.6 amole of alkaline phosphatase are required for each assay. The calculated K(i) for theophylline is 90 microM for the capillary electrophoretic enzyme-inhibitor assays.     

Flow injection electrochemical enzyme immunoassay for theophylline using a protein A immunoreactor and p-aminophenyl phosphate-p-aminophenol as the detection system.

A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass-protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V versus the saturated calomel electrode. For each sample the antibody-protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.     

Umbelliferone Phosphate as a Substrate for Acid and Alkaline Phosphatase

Abstract

The results of a complete study of 8 substrates for acid and alkaline phosphatase indicated 7-hydroxycoumarin (umbelliferone) phosphate to be the best substrate for the analysis of these enzymes. Using this ester, from 10^?6 to 2 × 10^?2 units per ml. of alkaline phosphatase and 10^?5 to 0.06 units per ml. of acid phosphatase can be determined with an accuracy and precision of about 1.5%. Samples of serum as small as 1 μ;1. can be assayed. Analysis is performed by a direct initial reaction rate method in 2–3 minutes.     

Immunosensor for the Determination of Okadaic Acid Based on Screen-Printed Electrode

A disposable immunosensor for okadaic acid (OA), using a screen-printed electrode (SPE), was developed and characterised. Detection of the product, p-aminophenol, resulting from the reaction catalysed by alkaline phosphatase (AP), was carried out using an amperometric three-electrode system poised at a voltage of + 300 mV versus Ag/AgCl. Alkaline phosphatase was used as a label for the antigen, OA, and two kinds of alkaline phosphatase preparation were studied for the conjugation of okadaic acid. The calibration curve for okadaic acid obtained from the conjugate created from low-activity AP, 969 units/mg, was unsatisfactory in terms of sensitivity, but a high-activity conjugate delivered the required sensitivity and limit of detection. Studies on the stability of the sensor with α-OA antibody and OA-AP conjugate showed that the current response decreased drastically after one day. Stabilisation strategies have been formulated to overcome this problem. The calibration curve obtained with the high activity conjugate was linear up to 40 ng/ml of okadaic acid with a minimum concentration of analyte detected of 5 ng/ml and a detection limit of 2 ng/ml.     

Highly sensitive assay for alkaline and acid phosphatase activities by high-performance liquid chromatography with electrochemical detection

A highly sensitive and specific assay for alkaline and acid phosphatases in biological materials, such as plasma and saliva, has been established. Phenol, formed enzymatically from the substrate phenylphosphate, was determined by high-performance liquid chromatography with electrochemical detection. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 5 pmol. Thus, as little as 0.5 microliter of rat plasma or 10 microliters of human saliva is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in saliva were found to be 1.12 +/- 0.12 nmol/min/ml and 9.79 +/- 1.23 nmol/min/ml, respectively. This new assay method should be applicable to extremely small biological samples.     

High-Sensitive Visually Controlled Membrane-Type Quantitation of NAD and Alkaline Phosphatase

Abstract

New high-sensitive visually controlled membrane-type analytical methods are proposed for quantitation of nicotineamide adenine dinucleotide and alkaline phosphatase in water solutions. The methods are based on using nitrocellulose membrane as a solid matrix on which the components of one-enzyme cofactor regeneration system are being immobilised by adsorption. In the presence of substances to be assayed, the end colored product is being adsorbed on the matrix as a result of enzymatic cyclic NAD/NADH regeneration in the active site of the matrix-bound alcohol dehydrogenase and some chemical successive reactions. Its colored intensity is a measure of the concentration of the analysed substances in solution. The general principle of NAD or alkaline phosphatase determination is successive immobilisation of separate components of the system (N-(6′-aminohexyl)salicylamide and horse liver alcohol dehydrogenase) on the matrix by adding their solutions to the wells of a specially designed cell with the membrane bottoms. In the case of alkaline phosphatase, the enzyme acted on NADPH as on a substrate. The reaction product, NAD was detected in the subsequent reaction of coenzyme regeneration. The other components of the amplifying system were added in substrate solutions at the stage of the alcohol dehydrogenase reaction. The lower detection limits for NAD and alkaline phosphatase were 3 × 10^?9 M and 1 × 10^?14 M respectively, the volume of the test sample ? 20 μl, the time of assay ? 5 min. The working concentration ranges were from 3 × 10^?9 to 1 × 10^?7 M and from 1 × 10^?14 to 1 × 10^?10 M levels for NAD(H) and alkaline phosphatase, respectively.     

Nanoporous gold as a solid support for protein immobilization and development of an electrochemical immunoassay for prostate specific antigen and carcinoembryonic antigen

Nanoporous gold (NPG) was utilized as a support for immobilizing alkaline phosphatase (ALP) conjugated to monoclonal antibodies against either prostate specific antigen (PSA) or carcinoembryonic antigen (CEA). The antibody-ALP conjugates were coupled to self-assembled monolayers of lipoic acid and used in direct kinetic assays. Using the enzyme substrate p-aminophenylphosphate, the product p-aminophenol was detected by its oxidation near 0.1?V (vs. Ag|AgCl) using square wave voltammetry. The difference in peak current arising from oxidation of p-aminophenol before and after incubation with biomarker increased with biomarker concentration. The response to these two biomarkers was linear up to 10?ng mL^?1 for CEA and up to 30?ng mL^?1 for PSA. The effect of interference on the PSA assay was studied using bovine serum albumin (BSA) as a model albumin protein. The effect of interference from a serum matrix was examined for the PSA assay using newborn calf serum. A competitive version of the immunoassay using antigen immobilized onto the NPG surface was highly sensitive at lower antigen concentration. Estimates of the surface coverage of the antibody-ALP conjugates on the NPG surface are presented.

High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenylmethylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-micron, 250 X 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile-water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile-methanol-tetrahydrofuran-water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (lambda max = 264) was about 5 ng/ml and by fluorescence detection (lambda excitation = 270 nm, lambda emission = 300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 +/- 4.2% from serum and 102.0 +/- 2.62% from urine.     

Small volume bead assay for ovalbumin with electrochemical detection

A bead based sandwich enzyme immunoassay coupled to electrochemical detection for ovalbumin has been developed. The enzyme label alkaline phosphatase was used to convert the substrate 4-aminophenyl phosphate to electroactive product 4-aminophenol. The detection was done in a microdrop by continuously monitoring the enzyme turnover with a rotating disk electrode. This reduces dilution of the enzyme product, a key to achieving low detection limits. The assay developed has a detection limit of 0.1 ng ml-1. Assay sensitivity in complex matrices such as food and serum was compared.     

Tyrosine decarboxylase

We have developed a highly sensitive and rapid spectrophotometric assay for tyrosine decarboxylase that can be applied to determining pyridoxal-5'-phosphate. In the assay, tyramine, a product of tyrosine decarboxylation, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a product soluble in toluene whereas tyrosine does not. We determined the amount of tyramine produced enzymatically by reading the absorbance at 340 nm of a toluene extract of the reaction mixture. This method is capable of detecting as low as 2.9 micrograms/mL of the enzyme. Using this method, we find the Km for tyrosine decarboxylase from Streptococcus faecalis to be 3.55 X 10(-4)M. We have also developed a specific and extremely sensitive method for determining pyridoxal-5'-phosphate, a cofactor of the enzyme, by using this spectrophotometric assay with apotyrosine decarboxylase.     

Amperometric immunosensor for detection of antibodies of Salmonella typhi in patient serum

An amperometric sensor for detection of antibodies to Salmonella typhi in the serum of patients was developed. This involved usage of screen-printed electrodes and recombinant flagellin fusion protein. An indirect enzyme-linked immunosorbant assay was used for detection of antibodies to S. typhi in the patient serum. The screen-printed electrodes were made using polystyrene and graphite. These electrodes were tested for their ability to detect 1-naphthol, which is the product formed due to the hydrolysis of the substrate 1-naphthyl phosphate by the enzyme alkaline phosphatase. These electrodes were coated with recombinant flagellin fusion protein made by recombinant DNA technology and blocked with bovine serum albumin (BSA). Further they were incubated with patient serum and goat anti-human alkaline phosphatase conjugate. The immunosensing was performed by using amperometric method. Pooled human serum samples from apparently healthy individuals were used as control. Both the pooled healthy human serum samples and patient sera were subjected to Widal agglutination test and amperometric method. A 100% correlation was found between the Widal test and amperometric method. The time taken for the detection by electrochemical method is 1 h and 15 min, while the time taken by Widal test is 18 h.     

Automated flow immunoassay for detection of food allergen using anion-exchange resin and alkaline phosphatase conjugated immunoglobulin G

A novel automated flow immunoassay system is developed for the detection of wheat protein as a food allergen. A polyclonal immunoglobulin G (IgG) antibody was isolated from serum of a rat with a wheat allergy, then reductively treated using dithiothreitol and covalently coupled to alkaline phosphatase. This conjugate was used in a non-competitive binding assay. The automated system was equipped with an immunoreaction column, an anion-exchange column, a luminescent reaction column and a photodetector. Antibody–antigen complexes were separated from their free conjugate on the basis of a difference in isoelectric point (pI) by an anion-exchange column. This simple technique permits the assay of wheat-protein allergen in 25 min with a reliable detection limit of 5 μg/ml. The anion-exchange column was regenerated by occasional elution with N-methylpiperazine buffer (pH 5.0) containing 0.5 M NaCl, to remove free conjugate. Free conjugate recovered in this manner could be reused up to four times without significant decrease in sensitivity of the immunoassay.     

Supramolecular Tandem Assay for Pyridoxal-5′-phosphate by the Reporter Pair of Guanidinocalix[5]Arene and Fluorescein

Guanidinocalix[5]arene and fluorescein reporter pair has been chosen to set up a supramolecular tandem assay principle based on the differential recognition of pyridoxal-5′-phosphate (the substrate of alkaline phosphatase, ALP), pyridoxal (the product of ALP) and phosphate (the product of ALP). This supramolecular tandem assay system offers an opportunity to monitor the activity of ALP in a label-free, continuous, and real-time manner. More importantly, a calibration curve can be given for selective and quantitative detection of pyridoxal-5′-phosphate (biomarker for several diseases).