DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

• MeSH
• DNA-ligasy chemie   MeSH
• DNA chemie   MeSH
• elektrochemické techniky přístrojové vybavení   metody   MeSH
• enzymatické testy přístrojové vybavení   metody   MeSH
• enzymy imobilizované chemie   MeSH
• Escherichia coli chemie   enzymologie   MeSH
• magnetismus MeSH
• proteiny z Escherichia coli chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Screen-printed platinum electrodes as transducer and magnetic beads as solid phase were combined to develop a particle-based electrochemical immunosensor for monitoring the serious food allergen ovalbumin. The standard arrangement of enzyme-linked immunosorbent assay became the basis for designing the immunosensor. A sandwich-type immunocomplex was formed between magnetic particles functionalized with specific anti-ovalbumin immunoglobulin G and captured ovalbumin molecules, and secondary anti-ovalbumin antibodies conjugated with the enzyme horseradish peroxidase were subsequently added as label tag. The electrochemical signal proportional to the enzymatic reaction of horseradish peroxidase during the reduction of hydrogen peroxide with thionine as electron mediator was measured by linear sweep voltammetry. The newly established method of ovalbumin detection exhibits high sensitivity suitable for quantification in the range of 11 to 222nM and a detection limit of 5nM. Magnetic beads-based assay format using external magnets for rapid and simple separation has been proven to be an excellent basis for electrochemical detection and quantification of food allergens in highly complex sample matrices.

• MeSH
• biosenzitivní techniky přístrojové vybavení   metody   MeSH
• elektrická vodivost MeSH
• elektrochemie MeSH
• elektrody MeSH
• imobilizační protilátky chemie   imunologie   MeSH
• imunoanalýza přístrojové vybavení   metody   MeSH
• limita detekce MeSH
• magnety chemie   MeSH
• mikrosféry * MeSH
• ovalbumin škodlivé účinky   analýza   MeSH
• platina chemie   MeSH
• potravinová alergie metabolismus   MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This review (with 129 refs) summarizes the progress in electrochemical immunoassays combined with magnetic particles that was made in the past 5 years. The specifity of antibodies linked to electrochemical transduction (by amperometry, voltammetry, impedimetry or electrochemiluminescence) gains further attractive features by introducing magnetic nanoparticles (MNPs). This enables fairly easy preconcentration of analytes, minimizes matrix effects, and introduces an appropriate label. Following an introduction into the fundamentals of electrochemical immunoassays and on nanomaterials for respective uses, a large chapter addresses method for magnetic capture and preconcentration of analytes. A next chapter discusses commonly used labels such as dots, enzymes, metal and metal oxide nanoparticles and combined clusters. The large field of hybrid nanomaterials for use in such immunoassays is discussed next, with a focus on MNPs composites with various kinds of graphene variants, polydopamine, noble metal nanoparticles or nanotubes. Typical applications address clinical markers (mainly blood and urine parameters), diagnosis of cancer (markers and cells), detection of pathogens (with subsections on viruses and bacteria), and environmental and food contaminants as toxic agents and pesticides. A concluding section summarizes the present status, current challenges, and highlights future trends. Graphical abstract Magnetic nanoparticles (MNP) with antibodies (Ab) capture and preconcentrate analyte from sample (a) and afterwards become magnetically (b) or immunospecifically (c) bound at an electrode. Signal either increases due to the presence of alabel (b) or decreases as the redox probe is blocked (c).

• MeSH
• elektrochemie metody   MeSH
• imunoanalýza metody   MeSH
• lidé MeSH
• magnety chemie   MeSH
• nanočástice chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Tumor Necrosis Factor Alpha (TNFα) is an important marker of inflammatory processes in human body. In the current healthcare, determination of TNFα blood or plasma level is done by Enzyme Linked Immuno-Sorbent Assay (ELISA) as a primary choice method. Piezoelectric immunosensors are analytical platform recording affinity interactions on their surface. It is inferred that the immunosensors would be a functional alternative to the ELISA. In this study, antibody against TNFα was immobilized on Quartz Crystal Microbalance (QCM) sensor and the same was made on magnetic particles. Human TNFα was measured in a way of interaction with QCM surface and then the particles were applied. The assay exerted sufficient limit of detection equal to 1.62pg/ml and it also fully correlated with standard ELISA tests. No interference by interleukin 6 or albumin was observed. Long term stability of the immunosensors lasting for at least three months was found. The immunosensors appears to be readily for practical performance and it would be an alternative to the standard ELISA especially when diagnoses made in field, homecare conditions or conditions of small hospitals as an emergency test.

• MeSH
• biosenzitivní techniky metody   MeSH
• elektřina * MeSH
• ELISA MeSH
• lidé MeSH
• mikrorovnovážné techniky křemenného krystalu * MeSH
• TNF-alfa analýza   MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• elektrochemické techniky MeSH
• ELISA metody   MeSH
• imunoenzymatické techniky * metody   MeSH
• lidé MeSH
• nádorové biomarkery * MeSH
• nanočástice * využití   MeSH
• Check Tag
• lidé MeSH

Guanosine derivatives are important for diagnosis of oxidative DNA damage including 8-hydroxy-2'-deoxyguanosine (8-OHdG) as one of the most abundant products of DNA oxidation. This compound is commonly determined in urine, which makes 8-OHdG a good non-invasive marker of oxidation stress. In this study, we optimized and tested the isolation of 8-OHdG from biological matrix by using paramagnetic particles with an antibody-modified surface. 8-OHdG was determined using 1-naphthol generated by alkaline phosphatase conjugated with the secondary antibody. 1-Naphthol was determined by stopped flow injection analysis (SFIA) with electrochemical detector using a glassy carbon working electrode and by stationary electrochemical detection using linear sweep voltammetry. A special modular electrochemical SFIA system which needs only 10  μL of sample including working buffer for one analysis was completely designed and successfully verified. The recoveries in different matrices and analyte concentration were estimated. Detection limit (3 S/N) was estimated as 5  pg/mL of 8-OHdG. This method promises to be very easily modified to microfluidic systems as "lab on valve". The optimized method had sufficient selectivity and thus could be used for determination of 8-OHDG in human urine and therefore for estimation of oxidative DNA damage as a result of oxidation stress in prostate cancer patients.

• MeSH
• alkalická fosfatasa MeSH
• deoxyguanosin analogy a deriváty   moč   MeSH
• elektrochemické techniky metody   MeSH
• ELISA metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• magnety MeSH
• mikrofluidní analytické techniky přístrojové vybavení   metody   MeSH
• mikrosféry MeSH
• nádory prostaty moč   MeSH
• naftoly MeSH
• oxidační stres MeSH
• protilátky MeSH
• průtoková injekční analýza MeSH
• robotika přístrojové vybavení   metody   MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Introduction: We aimed to compare the amniotic fluid interleukin (IL)-6 concentrations measured using the automated electrochemiluminescence immunoassay method and ELISA, and to establish an IL-6 concentration cut-off value for intra-amniotic inflammation (IAI) in preterm prelabor rupture of membranes (PPROM), which can be used in the automated electrochemiluminescence immunoassay method.Materials and methods: A total of 120 women with PPROM were included in this study. Amniotic fluid samples were obtained through transabdominal amniocentesis. IL-6 concentrations were assessed using both the automated electrochemiluminescence immunoassay method and ELISA, the current gold standard. IAI was defined as an amniotic fluid IL-6 concentration of ≥2600 pg/mL measured using ELISA.Results: A correlation between both assays was found (Spearman's rho = 0.97; p < .0001). Based on the receiver-operating characteristic curve for the identification of IAI (area under the curve = 0.99), a cut-off value of ≥3000 pg/mL was selected for the automated electrochemiluminescence immunoassay method with a sensitivity of 88%, specificity of 99%, positive predictive value of 97%, negative predictive value of 96%, and likelihood ratio of 76.Conclusions: For amniotic fluid IL-6 concentrations assessed using the automated electrochemiluminescence immunoassay method, a cut-off value of 3000 pg/mL was indicated for diagnosing IAI in women with PPROM.

• MeSH
• biologické markery metabolismus   MeSH
• chorioamnionitida diagnóza   etiologie   metabolismus   MeSH
• dospělí MeSH
• elektrochemické techniky metody   MeSH
• ELISA MeSH
• imunoanalýza metody   MeSH
• interleukin-6 metabolismus   MeSH
• lidé MeSH
• plodová voda metabolismus   MeSH
• předčasný odtok plodové vody patofyziologie   MeSH
• retrospektivní studie MeSH
• senzitivita a specificita MeSH
• těhotenství MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Antibodies are macromolecular structures with a high affinity to a molecule called the antigen. Though the natural aim of antibodies is the protection of the body from pathogens, they have been found to play a useful part in a number of specific treatments and diagnoses. This wider role for antibodies extends to assays where antibodies provide a recognition capability improving available physical methods. Photometrical methods such as ELISA or flow cytometry and electrochemical, optical or piezoelectric immunosensors (biosensors) are typical examples. The present review summarizes facts about the parameters and the production of antibodies. The structure of immunoglobulins, and the production and purification of monoclonal and polyclonal antibodies are described in four chapters. The review was written in order to collect the available knowledge on antibodies into one study improving orientation in this field for anyone wanting to construct immunoassays.

• MeSH
• adjuvancia imunologická terapeutické užití   MeSH
• biologické bojové látky MeSH
• biosenzitivní techniky metody   trendy   využití   MeSH
• chemické bojové látky izolace a purifikace   MeSH
• ELISA metody   trendy   využití   MeSH
• financování organizované MeSH
• imunoanalýza metody   trendy   využití   MeSH
• imunoglobuliny diagnostické užití   imunologie   izolace a purifikace   MeSH
• laboratorní zvířata MeSH
• lidé MeSH
• monoklonální protilátky diagnostické užití   imunologie   izolace a purifikace   MeSH
• protilátky diagnostické užití   imunologie   izolace a purifikace   MeSH
• tvorba protilátek MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Interferon gamma (IFNγ) is a cytokine and an immunochemical marker that can be used for revealing of infectious diseases and especially for distinguishing of viral and some types of bacterial infections. Blood tests for IFNγ are typically based on immunoassays like Enzyme-Linked Immunosorbent Assay (ELISA). In this paper, a biosensor working on the principle of quartz crystal microbalance (QCM) was developed as an alternative to the standard analytical methods for IFNγ. The biosensor contained antibodies against IFNγ immobilized on QCM and also on gold nanoparticles. A sandwich containing QCM, gold nanoparticles and IFNγ was formed and formation of the sandwich caused decrease of oscillation frequency. The assay exerted limit of detection 5.7 pg/ml for a sample sized 50 μl and one measuring cycle was finished within 90 min. The assay by biosensor fully correlated to standard ELISA. In a conclusion, the biosensor appears to be a fully applicable analytical tool for a simple assay of IFNγ. Overall simplicity and no special requirement on staff and equipment are the major advantages of the here presented assay.

• MeSH
• biosenzitivní techniky * MeSH
• imunoanalýza MeSH
• interferon gama MeSH
• kovové nanočástice * MeSH
• křemen MeSH
• lidé MeSH
• mikrorovnovážné techniky křemenného krystalu MeSH
• zlato MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH(4) silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.

• MeSH
• avidin metabolismus   MeSH
• barvení a značení metody   MeSH
• biotin chemie   metabolismus   MeSH
• dendrimery MeSH
• ELISA MeSH
• kinetika MeSH
• mikroskopie atomárních sil MeSH
• molekulární modely MeSH
• nanokompozity chemie   MeSH
• polyaminy chemická syntéza   chemie   MeSH
• povrchová plasmonová rezonance MeSH
• spektrofotometrie ultrafialová MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• stříbro chemie   MeSH
• transmisní elektronová mikroskopie MeSH
• Publikační typ
• práce podpořená grantem MeSH