Use of glucose-6-phosphate dehydrogenase as a new label for nucleic acid hybridization reactions

We describe here a sensitive new procedure for detecting DNA hybridization by dot blots. The method utilizes DNA or oligonucleotide probes labeled with biotin, sulfone, or haptens that can be detected by glucose-6-phosphate dehydrogenase (G6PDH) conjugates. Biotin labeling of DNA gave the best sensitivity. G6PDH activity was revealed by staining or by bioluminescence using an FMN oxidoreductase and a luciferase from Beneckea harveyi. Bioluminescent detection offered better sensitivity and faster revelation than the colorimetric assay and was found to be very useful in visualizing single mutations in human DNA after hybridization with an allele-specific biotinylated oligonucleotide probe. Revelation can be performed using a luminometer, photographic films, or a very sensitive video camera. The detection is limited by the nonspecific binding of the labeled reagent (streptavidin or antibodies). This limit is similar to that obtained with other nonisotopic labeling procedures, but our method is faster and several hybridization reactions can be performed on the same support.     

Amplified surface plasmon resonance immunosensor for interferon-gamma based on a streptavidin-incorporated aptamer

Interferon-gamma (IFN-γ) is associated with susceptibility to tuberculosis, which is a major public health problem worldwide. Although significant progress has been made with regard to the design of enzyme immunoassays for IFN-γ, this assay is still labor-intensive and time-consuming. We therefore designed a DNA aptamer hairpin structure for the detection of IFN-γ with high sensitivity and selectivity. A streptavidin DNA aptamer was incorporated into the IFN-γ binding aptamer probe for the amplified detection of the target molecules. Initially, the probe remained in the inactive configuration. The addition of IFN-γ induced the rearrangement of the aptamer structure, allowing the self-assembly of the active streptavidin aptamer conformation for the streptavidin molecular recognition. Under optimized conditions, the detection limit was determined to be 33 pM, with a dynamic range from 0.3 to 333 nM, both of which were superior to those of corresponding optical sensors. Because combined aptamers are composed of nucleic acids, this optical aptasensor provided the advantages of high sensitivity, simplicity, reusability, and no further labeling or sample pre-treatment.     

Comparison of two fluorescence immunoassay methods for the detection of endocrine disrupting chemicals in water

We describe two fluorescence immunoassays capable of detecting endocrine disrupting compounds in waste water. The first fluorescence method is a heterogeneous assay using total internal reflection fluorescence (TIRF) detection. The second method is a homogeneous assay that utilizes energy transfer (ETIA). Both fluorescence immunoassays are compared with respect to detection principle and ability to quantify the model analytes estrone, estradiol, and ethinylestradiol in a complex matrix regarding recovery rates and limits of detection. Calibrations were performed for the three analytes using both fluorescence methods. Limits of detection between 0.01 and 0.85 microg/l are achieved. In addition, measurements in synthetic waste water spiked with the analytes were performed. Both immunoassays allow the detection in waste water with recovery rates in the range of 70-112%.     

Assay of creatine kinase in microtiter plates using thio-NAD to allow monitoring at 405 nM

An assay system for creatine kinase using microtiter plates and a plate reader that records absorbancies at 405 nM has been devised. The system is an adaptation of well-established assays that couple creatine kinase with the reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase (G6PDH), to give a measurable increase in reduced pyridine nucleotide quantitated by absorbance at 340 nM. Two features of this system are modified for reading at 405 nM: (i) The thioamido derivative of NAD is used because its reduced form exhibits a substantial increase in absorbance at 405 nM, the most commonly available wavelength on microplate readers; and (ii) glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is used because it can reduce either NAD or NADP (unlike most other G6PDH enzymes, which require NADP), thus making it unnecessary to use the more expensive thio-NADP. The rate of thio-NAD reduction is linear with enzyme concentration and time over a 20-fold range of concentrations of purified creatine kinase, and the assay also works well with myogenic cells allowed to grow and differentiate in the 96-well plate in which the assay is performed. This system offers considerable savings in cells, time, and material in studies of muscle cell differentiation, for which creatine kinase levels are frequently measured. It also provides a potential method for the convenient and economical measurement of activities of many other enzymes that can be coupled to reduction of thio-NAD.     

Enzyme inhibitor screening using a homogeneous proximity-based immunoassay for estradiol

The authors have previously reported a homogeneous time-resolved fluorescence proximity immunoassay for estradiol. The assay was based on luminescence resonance energy transfer between a long lifetime fluorescent europium(III) chelate-dyed nanoparticle donor and a short lifetime, near-infrared fluorescent acceptor. The energy transfer prolonged the lifetime of the sensitized acceptor emission, and the fluorescence of the acceptor was measured using a time-resolved detection. The developed immunoassay was employed to screen inhibitors for enzyme 17beta-hydroxysteroid dehydrogenase type 1. The enzyme overexpressed in MCF-7 cells catalyzed a reversible conversion of estroneto17beta-estradiol. The inhibition efficiency of the tested molecule was obtained by comparing the final concentration of converted estradiol after 60 min of conversion reaction in a sample and in a conversion control not containing an inhibitor. The Zbeta factor calculated using the E2 concentrations of the homogeneous assay was 0.64, demonstrating a relatively good performance of the assay. The results from the homogeneous assay were comparable with the results obtained using radioactively labeled estrone as a substrate and high-performance liquid chromatography (HPLC) separation of estrone and converted estradiol after the enzyme reaction. Thus, this homogeneous assay can simplify the primary screening of potential new drug molecules by replacing a tedious radiometric HPLC method.     

Biotinyl-estradiol derivatives in enzyme immunoassays: structural requirements for optimal antibody binding

The use of the avidin/biotin complex in immunoassays is well documented. No comprehensive studies, however, are available on the structural requirements of the linkage between biotin and small molecules to get an optimal antigen-antibody interaction. We have synthesized seven different biotinylated estradiol derivatives. They were evaluated in an antibody- and in an antigen-immobilized enzyme immunoassay system. All three derivatives lacking a spacer group were useless for use in immunoassays, demonstrating the importance of a long distance between the biotin- and estradiol-moiety. In addition, the chemical structure of the linkage at the site of attachment to the steroid skeleton is very important for the antibody recognition: it may either be rigid but identical to that one used in the immunogen (6-carboxymethyloxime), or must be structurally flexible as exemplified by a 6-amido-linkage. A rigid structure (hydrazone) different from that of the immunogen absolutely prevents antibody binding.     

A universal competitive fluorescence polarization activity assay for S-adenosylmethionine utilizing methyltransferases

A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect S-adenosylhomocysteine (AdoHcy), a product of all S-adenosylmethionine (AdoMet)-utilizing methyltransferase reactions. We employed commercially available anti-AdoHcy antibody and fluorescein-AdoHcy conjugate tracer to measure AdoHcy generated as a result of methyltransferase activity. AdoHcy competes with tracer in the antibody/tracer complex. The release of tracer results in a decrease in fluorescence polarization. Under optimized conditions, AdoHcy and AdoMet titrations demonstrated that the antibody had more than a 150-fold preference for binding AdoHcy relative to AdoMet. Mock methyltransferase reactions using both AdoHcy and AdoMet indicated that the assay tolerated 1 to 3 microM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 muM AdoMet. To validate the assay's ability to quantitate methyltransferase activity, the methyltransferase catechol-O-methyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proof-of-principle experiments. A time- and enzyme concentration-dependent decrease in fluorescence polarization was observed in the COMT assay that was developed. The IC(50) value obtained using a selective COMT inhibitor was consistent with previously published data. Thus, this sensitive and homogeneous assay is amenable for screening compounds for inhibitors of methyltransferase activity.     

Fluorescence protection assay: a novel homogeneous assay platform toward development of aptamer sensors for protein detection

Development of novel aptamer sensor strategies for rapid and selective assays of protein biomarkers plays crucial roles in proteomics and clinical diagnostics. Herein, we have developed a novel aptamer sensor strategy for homogeneous detection of protein targets based on fluorescence protection assay. This strategy is based on our reasoning that interaction of aptamer with its protein target may dramatically increase steric hindrance, which protects the fluorophore, fluorescein isothiocyannate (FITC), labeled at the binding pocket from accessing and quenching by the FITC antibody. The aptamer sensor strategy is demonstrated using a model protein target of immunoglobulin E (IgE), a known biomarker associated with atopic allergic diseases. The results reveal that the aptamer sensor shows substantial (>6-fold) fluorescence enhancement in response to the protein target, thereby verifying the mechanism of fluorescence protection. Moreover, the aptamer sensor displays improved specificity to other co-existing proteins and a desirable dynamic range within the IgE concentration from 0.1 to 50 nM with a readily achieved detection limit of 0.1 nM. Because of great robustness, easy operation and scalability for parallel assays, the developed homogeneous fluorescence protection assay strategy might create a new methodology for developing aptamer sensors in sensitive, selective detection of proteins.     

Inactivation of glucose-6-phosphate dehydrogenase in solution by low- and high-frequency ultrasound

Kinetics of inactivation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in 0.1 M phosphate buffer (pH 7.4) within temperature range from 36 to 50 degrees C was studied comparatively under conditions of exposure of enzyme solution to low-frequency (LF, 27 kHz, 60 W/cm2) or high-frequency (HF, 880 kHz, 1.0 W/cm2) ultrasound (USD). Inactivation of G6PDH was characterized by effective first-order rate constants: (k(in)) total (summarized) inactivation; (k(in)*) thermal inactivation; and (k(in)(usd)) ultrasonic inactivation. Dilution of enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k(in) > k(in)*. The rate constants increased upon increasing the temperature. The Arrhenius plots of the temperature dependencies of k(in) and k(in) (usd) have a salient point at 44 degrees C. The activation energy (Eact) of the total inactivation of G6PDH was higher than Eact for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: Eact in case of inactivation with low-frequency ultrasound (LF-USD) was higher than in case of inactivation with high-frequency ultrasound (HF-USD). The rate of the ultrasonic induced inactivation of this enzyme substantially decreased in the presence of low concentrations of traps of radicals HO. (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of the G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in enzyme solutions exposed to USD.     

An aptamer-capture based chromogenic assay for thrombin

A simple chromogenic assay for human alpha thrombin is developed through aptamer affinity capture and a subsequent enzyme reaction. Thrombin is captured on the aptamer-modified magnetic beads, and catalyzes the conversion of chromogenic substrates to optically measured products. The measurement of the generated products by an absorbance spectrometer allows for the final quantification of thrombin. This assay shows high sensitivity by taking advantage of sample enrichment and enzyme amplification, and exhibits good specificity by involving the selective aptamer binding and the specific enzyme reaction. A concentration detection limit of 40 fM can be reached when the tripeptide substrate of tosyl-Gly-Pro-Arg-p-nitroanilide is used in a 24 h enzyme reaction, and the use of 2h enzyme reaction in the assay enables the detection of 400 fM thrombin for a rapid analysis. This assay can be applied to detect thrombin in dilute human serum.     

Estradiol assay by microtitre plate enzyme immunoassay

Development of a simple enzyme linked immunosorbent assay (ELISA) for estradiol in serum extracts is described. The assay involves use of a 96-well microtitre plate, designed for immunoassay, as the support for a purified, high-titre antiserum, raised against estradiol-6(O)-carboxymethyloxime linked to bovine serum albumin, and using horseradish peroxidase-labelled estradiol-6-(O)-carboxymethyloxime as the labelled species, with 2,2'-azino-bis-(3-ethylbenzthiazoline sulfonic acid) diammonium salt (ABTS) as the chromogenic substrate. The assay characteristics rival those of radio- or chemiluminescence immunoassays for estradiol.     

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.     

Bioluminescent assays using glucose-6-phosphate dehydrogenase: application to biotin and streptavidin detection

A streptavidin-glucose-6-phosphate dehydrogenase (G6PDH) conjugate was synthesized and its properties were studied, along with those of biotin-G6PDH conjugates. Two bioluminescent assays were used. Streptavidin was assayed in two steps: streptavidin samples were first incubated with a small amount of biotin-G6PDH and then with biotinylated rabbit gamma-globulins. The complex was immobilized on a bioluminescent immunoadsorbent. In the single-step biotin assay, free biotin was allowed to compete with biotin linked to rabbit gamma-globulins for binding to streptavidin-G6PDH in the presence of the bioluminescent immunoadsorbent. Neither assay required washing or separation steps and the sensitivity was 0.2 ng for streptavidin and 100 fg for biotin. Different applications are described: studies of biotin reactivity when linked to probes in solution or immobilized, and quantitation of biotin in biotinylated DNA probes and oligonucleotides.     

Monoclonal antibodies to glucose-6-phosphate dehydrogenase (G6PDH) form cyclic 1:1 complexes with G6PDH and act as regulatory subunits

A number of IgG monoclonal antibodies against L. mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) have been prepared. Four of the antibodies form 1:1 enzyme-antibody complexes which are stabilized in the presence of glucose-6-phosphate (G6P) and have greatly reduced enzyme activity. In the absence of G6P, the 1:1 complexes convert gradually to a more active multimeric form. Reduction of the IgG inter-heavy chain disulfides partially relieves inhibition and removes the G6P requirement for stability. F(ab')2 fragments of one of the antibodies behave similarly to the intact IgG. Reduction of the disulfides in the G6PDH-F(ab')2 complex leads to complete recovery of activity. The activity of complexes of G6PDH with reduced antibodies or Fab with digoxin bound to the antibody or Fab sulfhydryl groups can be modulated with antibodies to digoxin. The anti-G6PDH antibodies bridge two identical epitopes of this two subunit enzyme and simulate the function of regulatory subunits in which anti-digoxin acts as an activator. The system can be used to provide a sensitive homogeneous immunoassay for digoxin.     

A versatile graphene-based fluorescence "on/off" switch for multiplex detection of various targets

We have designed a versatile molecular beacon (MB)-like probe for the multiplex sensing of targets such as sequence-specific DNA, protein, metal ions and small molecule compounds based on the self-assembled ssDNA-graphene oxide (ssDNA-GO) architecture. The probe employs fluorescence "on/off" switching strategy in a single step in homogeneous solution. Compared to traditional molecular beacons, the proposed design is simple to prepare and manipulate and has little background interference, but still gives superior sensitivity and rapid response. More importantly, this ssDNA-GO architecture can serve as a universal beacon platform by simply changing the types of ssDNA sequences for the different targets. In this work, the ssDNA-GO architecture probe has been successfully applied in the multiplex detection of sequence-specific DNA, thrombin, Ag(+), Hg(2+) and cysteine, and the limit of detection was 1 nM, 5 nM, 20 nM, 5.7 nM and 60 nM, respectively. The results demonstrate that the ssDNA-GO architecture can be an excellent and versatile platform for sensing multiplex analytes, easily replacing the universal molecular beacon.     

Label-free homogeneous detection of immunoglobulin E by an aptameric enzyme subunit

We have developed an aptameric enzyme subunit (AES) for immunoglobulin E (IgE) sensing. AES is an artificial enzyme subunit constructed from two different aptamers and does not require any modification. Using the AES, the target molecule can be detected by measuring enzymatic activity in homogeneous solution. We connected IgE-binding aptamer and its complementary strand to split thrombin-inhibiting aptamer. The hybrid of these two oligonucleotides inhibited thrombin activity and it decreased in the presence of IgE. We were able to detect IgE by using this AES in homogeneous solution with a detection limit of 50 pmol.     

Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films

Summary The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters involved with the cytochemical assay as it is applied to sections or smears. The enzyme activity of G6PDH incorporated in the model films was also assayed biochemically. Optimal conditions for retaining the maximum amount of enzymic activity are described. The behaviour of G6PDH towards enzyme inhibitors was found to be similar in model films and in solution. With TNBT, absorbance measurements at a single wavelength (535 nm) were used to estimate the enzyme activity quantitatively. When carried out under standardized conditions, both the cytochemical and biochemical assay showed a linear relation with the time of incubation and obeyed the Beer-Lambert law. The correlation between biochemical and cytochemical data was very high, which enabled cytochemical data to be converted into absolute units of enzyme activity. The data obtained in this way closely resembled the data of enzyme activity calculated from the absorbance of formazan produced inside polyacrylamide model films and afterwards extracted into a suitable solvent.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

A fiber-optic microarray biosensor using aptamers as receptors

A fiber-optic biosensor using an aptamer receptor has been developed for the measurement of thrombin. An antithrombin DNA aptamer was immobilized on the surface of silica microspheres, and these aptamer beads were distributed in microwells on the distal tip of an imaging fiber. A different oligonucleotide bead type prepared using the same method as the aptamer beads was also included in the microwells to measure the degree of nonspecific binding. The imaging fiber was coupled to a modified epifluorescence microscope system, and the distal end of the fiber was incubated with a fluorescein-labeled thrombin (F-thrombin) solution. Nonlabeled thrombin could be detected using a competitive binding assay with F-thrombin. The aptamer beads selectively bound to the target and could be reused without any sensitivity change. The fiber-optic microarray system has a detection limit of 1 nM for nonlabeled thrombin, and each test can be performed in ca. 15 min including the regeneration time.