BACKGROUND: Total gentamicin is a sum of five congeners C1, C1a, C2, C2a and minor C2b, which differ from each other in their methylation on the purpurosamine ring. Liquid chromatography with mass detection (LC-MS/MS) and specified calibration material enables the concentration of total gentamicin and its individual congeners to be analysed. METHODS: 50 µL serum was precipitated with acetonitrile in the presence of 0.5 mol/L formic acid. A RP BEH C18 1.7 µm 2.1x50 mm column maintained at 30 °C and tobramicin as the internal standard were used. Mass detection was performed in positive electrospray. The gentamicin results were compared with fluorescence polarization immunoassay (FPIA) and chemiluminiscent microparticle immunoassay (CMIA). Passing-Bablock regression analysis and Bland-Altman analysis were used. RESULTS: Calibration curves for individual gentamicin congeners were linear with correlation coefficients between 0.997 and 0.998. Recovery was 91.6-102.0% and the coefficients of variation 1.4-8.4%. The total gentamicin concentration was compared with immunoassay FPIA (LC-MSgen = 0.9798xPFIAgen) and CMIA (LC MSgen = 0.9835xCMIAgen) both with significant correlation (p < 0.001). CONCLUSION: The LC-MS/MS method is fast and precise and can be applied to routine TDM in patients. Comparing it to immunoassays makes it possible to measure concentration of gentamicin congeners, which may be important in the case of their different pharmacokinetics.

• Klíčová slova
• Chemiluminiscence microparticle immunoassay, Fluorescence polarization immunoassay, LC-MS/MS, Therapeutic drug monitoring, Total gentamicin concentration,
• MeSH
• chromatografie kapalinová MeSH
• fluorescenční polarizační imunologické testy MeSH
• gentamiciny * MeSH
• imunoanalýza MeSH
• lidé MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• gentamiciny * MeSH

Trilobolide (Tb) is a pharmacologically interesting sesquiterpene lactone isolated from Laser trilobum (L.) Borkh. Structural relation to a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin bring promising prospects for Tb to be used in the development of new anti-cancer drugs. As long as there are still unanswered questions regarding its investigation, a need for novel analytical tools emerge. Since immunoassays serve as one of powerful tools within the investigation of natural products, the development of indirect competitive enzyme-linked immunosorbent assay (ELISA) utilizing coating based on avidin-biotin technology is described. In our set-up of ELISA, newly synthesized biotinylated Tb served as immobilized competitor. Tb-carboxymethyloxime-bovine serum albumin (BSA) and Tb-succinoyl-BSA conjugates were used separately for immunization of rabbits. Two sets of polyclonal antibodies (RAbs) were obtained. Antibodies against Tb-succinoyl-BSA conjugate (RAb No. 206) were chosen as the best. Under optimized conditions, limit of detection and 50% intercept of our ELISA were 849pg/mL and 8.89ng/mL, respectively. The cross-reactivity (CR) was tested on 10 structurally related compounds and CR did not exceed 6.1%. The reproducibility of the system is expressed as intra- and inter-assay coefficients of variation (9.7% and 11.4%, respectively). Based on conducted experiments, we proposed the use of ELISA for quantification of Tb in complex biological matrices such as plant extracts. A method was applied to analyze three extracts obtained from different parts of L. trilobum. Data obtained were compared to those acquired by UHPLC-MS/MS. The concordance between the methods (103-87%) showed the ability of ELISA to quantify Tb.

• Klíčová slova
• Avidin-biotin, ELISA, Immunoassay, Laser trilobum, Synthesis, Trilobolide,
• MeSH
• Apiaceae chemie   MeSH
• butyráty analýza   MeSH
• ELISA MeSH
• furany analýza   MeSH
• imunoanalýza metody   MeSH
• králíci MeSH
• molekulární struktura MeSH
• protilátky imunologie   MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• butyráty MeSH
• furany MeSH
• protilátky MeSH
• trilobolide MeSH Prohlížeč

OBJECTIVE: The aim of this study was to compare results of two commercially available kits used for routine detection of Rotavirus A in human stool samples with results of commercial quantitative reverse-transcription PCR (RT-qPCR) test and in-house RT-qPCR. MATERIAL AND METHODS: In total, 749 stool samples were screen-ed with the use of four different methods. The samples were collected from four diagnostic laboratories from March 2016 to June 2017. Diagnose of gastrointestinal disorders was stated in one third of tested patients, the rest of samples was collected from patients with other primary diagnose. The samples were tested with the enzymatic immunoassay (EIA) (RIDASCREEN® Rotavirus) and with rapid diagnostic immunochromatographic test (RDT) (IMMUNOQUICK® No-Rot-Adeno). As a reference method a commercial RT-qPCR test was used (Primerdesign Genesig® Kit) and it was compared with in-house RT-qPCR test prepared in our laboratory. The samples which in the reference RT-qPCR gave positive signal of reaction in cycle 28 or higher (Ct 28) were assessed as negatives in order to include only samples with some clinical relevance into sensitivity determination. RESULTS: Diagnostic sensitivity was assessed as 84.2% for EIA and 82.5% for RDT. The specificity of those tests was calculated as 97.8% for EIA and 96.4% for RDT. The performance of both diagnostic tests describing their positive predictive value was determined to be 87.3% for EIA and 80.3% for RDT. Negative predictive value was calculated to be 97.2% for EIA and 96.8% for RDT. Proportion of RVA-positive samples determined with the reference RT-qPCR test with our own cut-off level was 15.2% (n=114). Comparisons of the in-house and reference RT-qPCR tests showed very good agreement of results. The sensitivity of the in-house test was 100% and its specificity 99.7%. CONCLUSIONS: RT-qPCR is more sensitive for surveillance of rotavirus gastroenteritis than routinely used EIA or RDT methods. The specificity of both evaluated tests was very high. However, EIA was in all performance parameters assessed better than RDT.

• Klíčová slova
• rotavirus A - enzymatic immunoassay - immunochromato-graphic test - RT-qPCR, rotavirus A - enzymatic immunoassay - immunochromato-graphic test - RT-qPCR.,
• MeSH
• chromatografie * normy   MeSH
• feces virologie   MeSH
• gastrointestinální nemoci diagnóza   virologie   MeSH
• imunoanalýza normy   MeSH
• imunoenzymatické techniky * normy   MeSH
• lidé MeSH
• polymerázová řetězová reakce s reverzní transkripcí * normy   MeSH
• rotavirové infekce * MeSH
• Rotavirus * izolace a purifikace   MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Over the last two decades, the group of techniques called affinity probe CE has been widely used for the detection and the determination of several types of biomolecules with high sensitivity. These techniques combine the low sample consumption and high separation power of CE with the selectivity of the probe to the target molecule. The assays can be defined according to the type of probe used: CE immunoassays, with an antibody as the probe, or aptamer-based CE, with an aptamer as the probe. Immunoassays are generally divided into homogeneous and heterogeneous groups, and homogeneous variant can be further performed in competitive or noncompetitive formats. Interacting partners are free in solution at homogeneous assay, as opposed to heterogeneous analyses, where one of them is immobilized onto a solid support. Highly sensitive fluorescence, chemiluminescence or electrochemical detections were typically used in this type of study. The use of the aptamers as probes has several advantages over antibodies such as shorter generation time, higher thermal stability, lower price, and lower variability. The aptamer-based CE technique was in practice utilized for the determination of proteins in biological fluids and environmentally or clinically important small molecules. Both techniques were also transferred to microchip. This review is focused on theoretical principles of these techniques and a summary of their applications in research.

• Klíčová slova
• Application, Aptamer, CE, Immunoassay, Microchip,
• MeSH
• antibakteriální látky analýza   MeSH
• aptamery nukleotidové * MeSH
• elektroforéza kapilární * MeSH
• imunoanalýza * MeSH
• laboratoř na čipu MeSH
• lidé MeSH
• protilátky krev   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• antibakteriální látky MeSH
• aptamery nukleotidové * MeSH
• protilátky MeSH

Surface engineering of upconverting nanoparticles (UCNPs) is crucial for their bioanalytical applications. Here, an antibody specific to cardiac troponin I (cTnI), an important biomarker for acute myocardial infection, was covalently immobilized on the surface of UCNPs to prepare a label for the detection of cTnI biomarker in an upconversion-linked immunoassay (ULISA). Core-shell UCNPs (NaYF4:Yb,Tm@NaYF4) were first coated with poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) and then conjugated to antibodies. The morphology (size and uniformity), hydrodynamic diameter, chemical composition, and amount of coating on the of UCNPs, as well as their upconversion luminescence, colloidal stability, and leaching of Y3+ ions into the surrounding media, were determined. The developed ULISA allowed reaching a limit of detection (LOD) of 0.13 ng/ml and 0.25 ng/ml of cTnI in plasma and serum, respectively, which represents 12- and 2-fold improvement to conventional enzyme-linked immunosorbent based on the same immunoreagents.

• Klíčová slova
• Bioconjugation, Cardiac troponin I, Immunoassay, Photon-upconversion nanoparticle, Poly(methyl vinyl ether‐alt‐maleic acid), Upconversion-linked immunosorbent assay,
• MeSH
• imunoanalýza metody   MeSH
• limita detekce MeSH
• luminiscence MeSH
• nanočástice * chemie   MeSH
• troponin I analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• troponin I MeSH

We present a simple and fast one-step heterogeneous immunoassay, with performance characteristics that can enable easy and versatile adaptation to miniaturized, automated point-of-care systems. This novel analytical method uses magnetic and fluorescent beads as capture and detection agents respectively. Its main feature is the measurement of the fluorescent signal in the bound-free phase for (semi-)quantitative detection of analytes. Thus, no washing is required and the workflow consists only of sample and reagent supply, incubation, separation and detection. The immunoassay concept is demonstrated with C-reactive protein (CRP), a systemic inflammation marker. CRP in only 5 μL of undiluted serum was measured in the range 20-140 mg L-1 (includes clinically relevant cut-off values). The limit of detection (LOD) was 22.1 ± 6.3 mg L-1 (incubation 15 min). A CRP certified reference material was measured on five different days. Intra- and inter-assay coefficients of variation were 4.6 ± 1.9% and 5.6% respectively. To demonstrate the compatibility of the assay concept with additional matrices and concentration ranges, three oral inflammation markers, namely matrix metalloproteinases 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1), were measured in saliva in the ranges 0.47-30 ng mL-1 for MMP-8 and MMP-9, and 0.69-44 ng mL-1 for TIMP-1. LODs were 0.24 ng mL-1, 0.38 ng mL-1 and 0.39 ng mL-1 respectively (incubation 20 min). Multiplexing capacity of the assay concept was also shown with these markers. The demonstrated excellent reproducibility of the results, combined with the versatility and low complexity of the introduced immunoassay concept, make it an attractive candidate for applied analytical chemistry and automated point-of-care testing.

• Klíčová slova
• Bound-free phase, Immunoassay, Inflammation, Micro/nanoparticles, Oral biomarkers, Saliva,
• MeSH
• C-reaktivní protein * MeSH
• imunoanalýza MeSH
• limita detekce MeSH
• reprodukovatelnost výsledků MeSH
• vyšetření u lůžka * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• C-reaktivní protein * MeSH

In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 µL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15-115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications.

• Klíčová slova
• bound-free phase, centrifugal microfluidics, immunoassay, inflammation, micro/nanoparticles, point-of-care, reagent storage,
• MeSH
• C-reaktivní protein * MeSH
• imunoanalýza metody   MeSH
• indikátory a reagencie MeSH
• lidé MeSH
• mikrofluidika * MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• C-reaktivní protein * MeSH
• indikátory a reagencie 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.

• Klíčová slova
• Bioassay, Biosensor, Cytokine, Diagnosis, ELISA, Immunoassay, Immunosensor, Infection, Lymphocyte, Macrophage, Virus,
• 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
• Názvy látek
• interferon gama MeSH
• křemen MeSH
• zlato MeSH

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H2O2/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H2O2 and o-phenylendiamine as substrates. Recovery of the assay was 97-112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.

• MeSH
• analýza potravin MeSH
• imunoenzymatické techniky MeSH
• indikátory a reagencie MeSH
• kolorimetrie metody   MeSH
• kur domácí MeSH
• luminiscenční měření MeSH
• muramidasa analýza   MeSH
• senzitivita a specificita MeSH
• vejce * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• indikátory a reagencie MeSH
• muramidasa MeSH

An electrochemical immunoassay for neopterin was developed using recently produced specific antibodies immobilized to protein A-coated magnetic beads in combination with differential pulse voltammetry and screen-printed array of electrodes. Neopterin-alkaline phosphatase conjugate was used as label in a competitive assay format. Multiplexed analysis of neopterin was demonstrated by replacing the traditional ELISA with electrochemical detection and the traditional plastic wells with screen-printed array of electrodes. The optimized electrochemical method, based on polyclonal antibodies, reached a limit of detection of 0.008 ng/mL with an average RSD %=10. Serum samples collected from patients with sepsis, healthy volunteers and other patients without a confirmed clinical diagnosis were also analyzed. The obtained results, compared with those of a commercial ELISA kit, had a significant correlation, showing the possibility to distinguish among the serum samples from ill or healthy subjects.

• Klíčová slova
• Electrochemical immunoassay, Magnetic beads, Neopterin, Screen-printed arrays, Serum samples,
• MeSH
• biologické markery MeSH
• biosenzitivní techniky * MeSH
• elektrochemické techniky MeSH
• elektrody MeSH
• imobilizované proteiny imunologie   MeSH
• imunoanalýza MeSH
• lidé MeSH
• neopterin analýza   krev   imunologie   MeSH
• protilátky imunologie   MeSH
• sepse krev   MeSH
• zánět MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• imobilizované proteiny MeSH
• neopterin MeSH
• protilátky MeSH