المؤلفون: Patrick Valere Tsouh Fokou, Brice Mariscal Tchatat Tali, Darline Dize, Cedric Derick Jiatsa Mbouna, Cyrille Armel Njampa Ngansop, Rodrigue Keumoe, Lauve Rachel Yamthe Tchokouaha, Jean Claude Tchouankeu, Fanny Escudie, James Duffy, Fabrice Fekam Boyom

المصدر: Analytical biochemistry. 648

مصطلحات موضوعية: Plasmodium, L-Lactate Dehydrogenase, Plasmodium falciparum, Biophysics, Drug Evaluation, Preclinical, Reproducibility of Results, Cell Biology, Microbial Sensitivity Tests, Mycobacterium tuberculosis, Biochemistry, Antimalarials, Humans, Colorimetry, Malaria, Falciparum, Molecular Biology

الوصف: Antimalarial drug discovery has been facilitated by the development of various in vitro drug susceptibility testing methods suitable for medium-throughput or high-throughput campaigns. Among many, the Plasmodium falciparum lactate dehydrogenase (PfLDH) assay has acceptable demand on equipment, labour, technical skills and affordability and offers a good opportunity for scientists in low- and middle-income countries to participate in the global effort of discovering future antimalarial drugs. Hence, to enable our search for novel antimalarial drugs, we implemented and examined assay conditions and validated the PfLDH-based method in our laboratory using a reference set of standard antimalarial drugs with known activity against Plasmodium falciparum strains. The PfLDH assay revealed acceptable linearity profiles of R

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::906c7a051c660d0c864ee894105b16e7Test
https://pubmed.ncbi.nlm.nih.gov/35321819Test

المؤلفون: Armin Nourani, David W. Wright, Christine F. Markwalter, Andrew G. Kantor

المصدر: Analytical Biochemistry. 612:114020

مصطلحات موضوعية: Nitrilotriacetic Acid, Aptamer, Plasmodium falciparum, Protozoan Proteins, Biophysics, Antigens, Protozoan, Sensitivity and Specificity, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Limit of Detection, Lactate dehydrogenase, Malaria elimination, parasitic diseases, medicine, Humans, Magnetite Nanoparticles, Molecular Biology, 030304 developmental biology, Detection limit, 0303 health sciences, Chromatography, L-Lactate Dehydrogenase, biology, Diagnostic Tests, Routine, Chemistry, 010401 analytical chemistry, Diagnostic test, Cell Biology, Aptamers, Nucleotide, medicine.disease, Malaria, 0104 chemical sciences, Kinetics, Zinc, biology.protein, Biomarker (medicine), Reagent Kits, Diagnostic, Antibody, Plasmodium vivax, Biomarkers, Protein Binding

الوصف: Rapid diagnostic tests (RDTs) are critical to the success of malaria elimination campaigns. These tests are rapid, user-friendly, and field-deployable to resource-limited regions. However, RDTs demonstrate poor sensitivity because they can only tolerate a small (5 μL) volume of blood, which limits the amount of protein biomarker delivered to the test. We have developed the An tibody-free D ual-biomarker R apid E nrichment W orkflow (AnDREW) for purifying histidine-rich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (PLDH) from large volume (150 μL) blood samples. We used Zn(II)NTA and aptamer-conjugated magnetic beads to capture HRP2 and PLDH, respectively. Both biomarkers were then eluted into RDT-compatible volumes using ethylene diamine tetraacetic acid (EDTA). We optimized both bead conjugates individually by enzyme-linked immunosorbent assays (ELISAs) and then combined the optimized capture and elution assays for both biomarkers to produce the AnDREW. The AnDREW-enhanced RDTs exhibited a 11-fold and 9-fold improvement in analytical sensitivity for detection of HRP2 and PLDH, respectively, when compared to unenhanced RDTs. Moreover, the limit of detection for PLDH was improved 11-fold for the AnDREW-enhanced RDTs (3.80 parasites/μL) compared to unenhanced RDTs (42.31 parasites/μL). Importantly, the AnDREW utilizes a pan-specific PLDH aptamer and improves upon existing methods by eluting both biomarkers without complexed antibodies.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2a6004ef720a489cb2200042f93d4863Test
https://doi.org/10.1016/j.ab.2020.114020Test

المؤلفون: Sara Samuei, Ashkan Shomali, Jila Fakkar, Biuck Habibi, Zolfaghar Rezvani

المصدر: Analytical Biochemistry. 521:31-39

مصطلحات موضوعية: Thermogravimetric analysis, Materials science, Biophysics, Analytical chemistry, Biosensing Techniques, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Biochemistry, Nanocomposites, law.invention, X-Ray Diffraction, law, Quantum Dots, Hydroxides, Humans, Fourier transform infrared spectroscopy, Electrodes, Molecular Biology, Nanocomposite, L-Lactate Dehydrogenase, Graphene, Electrochemical Techniques, Cell Biology, Chronoamperometry, 021001 nanoscience & nanotechnology, Carbon, 0104 chemical sciences, Glucose, Metals, Quantum dot, Electrode, Microscopy, Electron, Scanning, Graphite, Cyclic voltammetry, 0210 nano-technology

الوصف: In the present work, a novel nanocomposite based on the graphene quantum dots and CoNiAl-layered double-hydroxide was successfully synthesized by co-precipitation method. To achieve the morphological, structural and compositional information, the resulted nanocomposite was characterized by scanning electron microscopy X-ray diffraction, thermal gravimetric analysis, Fourier transform infrared spectroscopy, and photoluminescence. Then, the nanocomposite was used as a modifier to fabricate a modified carbon paste electrode as a non-enzymatic sensor for glucose determination. Electrochemical behavior and determination of glucose at the nanocomposite modified carbon paste electrode were investigated by cyclic voltammetry and chronoamperometry methods, respectively. The prepared sensor offered good electrocatalytic properties, fast response time, high reproducibility and stability. At the optimum conditions, the constructed sensor exhibits wide linear range; 0.01–14.0 mM with a detection limit of 6 μM (S/N = 3) and high sensitivity of 48.717 μAmM−1. Finally, the sensor was successfully applied to determine the glucose in real samples which demonstrated its applicability.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a7f8c59ea07bee4ecfe4deed614919c8Test
https://doi.org/10.1016/j.ab.2017.01.005Test

المؤلفون: Kuang-Pin Hsiung, Min-Chi Lan, Wei-Feng Chang, Chia-Chi Lin, Wei-Shiang Lai, Shu-Chen Kan, Yung-Chuan Liu, Chwen-Jen Shieh

المصدر: Analytical Biochemistry. 471:61-66

مصطلحات موضوعية: Biophysics, Tetrazolium Salts, Nicotinamide adenine dinucleotide, Biochemistry, chemistry.chemical_compound, Limit of Detection, Lactate dehydrogenase, Diaphorase, Animals, Humans, Lactic Acid, Molecular Biology, Reagent Strips, Detection limit, Chromatography, L-Lactate Dehydrogenase, Cell Biology, Hydrogen-Ion Concentration, Enzymes, Immobilized, NAD, Clostridium kluyveri, Lactic acid, Kinetics, chemistry, Rabbits, NAD+ kinase, Formazan, Quantitative analysis (chemistry)

الوصف: In this study, a dry assay of l -lactate via the enzymatic chromatographic test (ECT) was developed. An l -lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2 −6 U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l -lactate dehydrogenase (1 μl, 0.25 U/μl), and NAD + (2 μl, 1.5 × 10 −5 M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10 mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5 mM with a detection limit of 0.047 mM. This quantitative analysis process for l -lactate was easy to operate with good stability and was proper for the point-of-care testing applications.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::85ea2134c298e3ddd8f08dbf8b943fa2Test
https://doi.org/10.1016/j.ab.2014.11.015Test

المؤلفون: Gomez Perez, Mariela¹ gomez_perez.mariela@courrier.uqam.ca, Fourcade, Lyvia¹ lyvia.fourcade@gmail.com, Mateescu, Mircea Alexandru¹ mateescu.m-alexandru@uqam.ca, Paquin, Joanne¹ paquin.joanne@uqam.ca

المصدر: Analytical Biochemistry. Oct2017, Vol. 535, p43-46. 4p.

مصطلحات موضوعية: *MYOBLAST transfer therapy, *COPPER compounds, *L-Lactate dehydrogenase, *ENZYME activation, *NEURONS

مستخلص: Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea 2 , Cu(II)Ser 2 and CuCl 2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity. [ABSTRACT FROM AUTHOR]

المؤلفون: Keersten M. Davis, Christine F. Markwalter, David W. Wright

المصدر: Analytical biochemistry. 493

مصطلحات موضوعية: Immunomagnetic capture, Plasmodium falciparum, Biophysics, 02 engineering and technology, Immunomagnetic separation, 01 natural sciences, Biochemistry, Colorimetry (chemical method), chemistry.chemical_compound, Limit of Detection, Lactate dehydrogenase, parasitic diseases, Humans, Activity assay, Malaria, Falciparum, Molecular Biology, Whole blood, Enzyme Assays, Detection limit, Chromatography, biology, L-Lactate Dehydrogenase, Immunomagnetic Separation, Plasmodium lactate dehydrogenase, 010401 analytical chemistry, Cell Biology, 021001 nanoscience & nanotechnology, biology.organism_classification, Molecular biology, Enzyme assay, Malaria, 0104 chemical sciences, 3. Good health, chemistry, biology.protein, Colorimetry, Antibody, 0210 nano-technology

الوصف: We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/μl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4–6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0eed1b4d67a48558a5666a947e13ddc1Test
https://pubmed.ncbi.nlm.nih.gov/26475567Test

المؤلفون: Osamu Mori, Yoshimichi Kozuka, Yuji Sakasegawa, Mineo Yamazaki, Takuya Ohkubo, Kiyotoshi Kaneko, Naomi S. Hachiya, Hidehiro Mizusawa

المصدر: Analytical Biochemistry. 347:106-111

مصطلحات موضوعية: Male, Protein Denaturation, Protein Folding, Saccharomyces cerevisiae Proteins, Immunoblotting, Biophysics, Sensitivity and Specificity, Biochemistry, Inclusion bodies, chemistry.chemical_compound, Pick Disease of the Brain, Immunoblot Analysis, Lactate dehydrogenase, Humans, Guanidine, Molecular Biology, Actin, Aged, Inclusion Bodies, L-Lactate Dehydrogenase, Lasers, Cell Biology, chemistry, Unfolded protein response, D-lactate dehydrogenase, Urea, Electrophoresis, Polyacrylamide Gel, Female, L-Lactate Dehydrogenase (Cytochrome), Microdissection

الوصف: We established a histobiochemical approach targeting micron-order inclusion bodies possessing extensive aggregation properties in situ by using a nonchemical denaturant (oligomeric actin interacting protein 2/ d -lactate dehydrogenase protein 2 [Aip2p/Dld2p]) with the combinatorial method of laser-microdissection and immunoblot analysis. As a model, pick bodies were chosen and laser-microdissected from three different brain regions of two patients with Pick’s disease. Initially, 500 to 2000 pick bodies were applied onto SDS–PAGE gels after boiling in Laemmli’s sample buffer according to established immunoblotting procedures; however, only faint signals were obtained. Following negative results with chemical denaturants or detergent, including 6 M guanidine hydrochloride, 8 M urea, and 2% SDS, the laser-microdissected pick bodies were pretreated with oligomeric Aip2p/Dld2p, which possesses robust protein unfolding activity under biological conditions. Strikingly, only one pick body was sufficient to illustrate an immunoblot signal, indicating that pretreatment with oligomeric Aip2p/Dld2p enhanced the immunoblot sensitivity by more than 100-fold. Pretreatment with oligomeric Aip2p/Dld2p also allowed us to quantify the total protein content of pick bodies. Thus, use of oligomeric Aip2p/Dld2p significantly contributed toward the acquisition of information pertaining to the molecular profile of proteins possessing an extensive aggregation property, particularly in small amounts.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::627892b31ee1404df899e21e20fbc9ceTest
https://doi.org/10.1016/j.ab.2005.09.004Test

المؤلفون: Anja-K. Münster-Kühnel, Rita Gerardy-Schahn, Akiko Fujita, Chihiro Sato, Ken Kitajima

المصدر: Analytical Biochemistry. 337:12-21

مصطلحات موضوعية: Cytidine monophosphate, Biophysics, Nicotinamide adenine dinucleotide, Biochemistry, Photometry, Mice, chemistry.chemical_compound, Lactate dehydrogenase, Methods, Animals, Molecular Biology, chemistry.chemical_classification, N-Acylneuraminate Cytidylyltransferase, L-Lactate Dehydrogenase, biology, Cell Biology, NAD, Lyase, Molecular biology, N-Acetylneuraminic Acid, Enzyme assay, Sialic acid, Enzyme, chemistry, biology.protein, NAD+ kinase

الوصف: A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::902e844971ce864bac25fed84c85b12cTest
https://doi.org/10.1016/j.ab.2004.10.033Test

المؤلفون: Maxim Zakhartsev, Hans O. Pörtner, Ronny Blust

المصدر: Analytical biochemistry
EPIC3Analytical Biochemistry, 330, pp. 10-20

مصطلحات موضوعية: 030310 physiology, Biophysics, Analytical chemistry, Activation energy, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, symbols.namesake, Lactate dehydrogenase, Spectrophotometry, medicine, Animals, Gadus, Muscle, Skeletal, Molecular Biology, 030304 developmental biology, Arrhenius equation, 0303 health sciences, Temperature control, L-Lactate Dehydrogenase, biology, medicine.diagnostic_test, Cell Biology, biology.organism_classification, Microplate Reader, Freezing point, Cold Temperature, Kinetics, Gadus morhua, chemistry, symbols, Thermodynamics

الوصف: Analyses of temperature-dependent kinetic parameters in enzymes extracted from tissues of ectothermic animals are usually carried out within the range of physiological temperatures (0-40 degrees C). However, multisample spectrophotometers (so-called microplate readers) with efficient wide-range temperature control (including cooling) have previously been unavailable. This limits the statistical quality of the measurements. A temperature-controlled microplate was designed for a 96-well microplate reader to overcome this limitation. This so-called T-microplate is able to control assay temperature between the freezing point of a liquid sample and 60 degrees C with high stability and accuracy in any data acquisition mode. At 4 degrees C the accuracy of the temperature control was +/-0.1 degrees C and temperature homogeneity across the microplate was +/-0.3 degrees C. As examples, analyses of the temperature dependence of Michaelis-Menten (K'(PYR)(m) and substrate inhibition (K'(PYR)(si) constants for pyruvate, of the maximal rate of reaction (V'(max), of the apparent Arrhenius activation energy (E(A), and of the Gibbs free-energy change (deltaG) of lactate dehydrogenases from muscle of Atlantic cod Gadus morhua acclimated to 4 degrees C are described. The large dataset obtained allowed evaluation of a new mechanism of metabolic compensation in response to seasonal temperature change.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f879837c6a1e331b058984fed0e3818aTest
https://doi.org/10.1016/j.ab.2004.03.070Test

المؤلفون: Catherine J. Pachuk, Meiqing Lu, Manoj Samuel, Chandrasekha Satishchandran

المصدر: Analytical Biochemistry. 313:301-306

مصطلحات موضوعية: DNA, Bacterial, Pyruvate Kinase, Biophysics, Sensitivity and Specificity, Biochemistry, Substrate Specificity, chemistry.chemical_compound, Adenosine Triphosphate, RNA, Transfer, Lactate dehydrogenase, Escherichia coli, Magnesium, Molecular Biology, chemistry.chemical_classification, Deoxyribonucleases, Chromatography, DNA clamp, L-Lactate Dehydrogenase, Hydrolysis, Substrate (chemistry), Deoxyribonuclease, Cell Biology, NAD, Adenosine Diphosphate, Kinetics, Enzyme, chemistry, Spectrophotometry, Phosphodiester bond, NAD+ kinase, DNA, Circular, Oxidation-Reduction, DNA, Plasmids

الوصف: A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::02f1b85041de42dc2d5abb4a2612d293Test
https://doi.org/10.1016/s0003-2697Test(02)00591-2