Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1-14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.
- MeSH
- europium chemie MeSH
- hmotnostní spektrometrie MeSH
- imunoblotting MeSH
- kadmium chemie MeSH
- kovové nanočástice chemie MeSH
- kvantové tečky chemie MeSH
- lasery MeSH
- lidé MeSH
- limita detekce MeSH
- monoklonální protilátky chemie farmakologie MeSH
- nádorový supresorový protein p53 antagonisté a inhibitory MeSH
- stříbro chemie MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Dairy cow feed contains, among other ingredients, soybeans, legumes, and clover, plants that are rich in phytoestrogens. Several publications have reported a positive influence of phytoestrogens on human health; however, several unfavorable effects have also been reported. In this work, a simple, selective, and eco-friendly method of phytoestrogen isolation based on the technique of noncovalent molecular imprinting was developed. Genistein was used as a template, and dopamine was chosen as a functional monomer. A layer of molecularly imprinted polymers was created in a microtitration well plate. The binding capability and selective properties of obtained molecularly imprinted polymers were investigated. The imprinted polymers exhibited higher binding affinity toward chosen phytoestrogen than did the nonimprinted polymers. A selectivity factor of 6.94 was calculated, confirming satisfactory selectivity of the polymeric layer. The applicability of the proposed sensing method was tested by isolation of genistein from a real sample of bovine milk and combined with micellar electrokinetic capillary chromatography with UV-visible detection.
In this paper, we describe the labelling of antibodies by gold nanoparticles (AuNPs) with diameters of 10 and 60 nm with detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Additionally, the AuNPs labelling strategy is compared with commercially available labelling reagents based on MeCAT (metal coded affinity tagging). Proof of principle experiments based on dot blot experiments were performed. The two labelling methods investigated were compared by sensitivity and limit of detection (LOD). The absolute LODs achieved were in the range of tens of picograms for AuNP labelling compared to a few hundred picograms by the MeCAT labelling.
- MeSH
- hmotnostní spektrometrie metody MeSH
- imunoanalýza metody MeSH
- indikátory a reagencie chemie MeSH
- kovové nanočástice chemie MeSH
- lasery * MeSH
- limita detekce MeSH
- ověření koncepční studie MeSH
- protilátky chemie MeSH
- specificita protilátek MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
For the first time, the combination of molecularly imprinted polymer (MIP) technology with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is presented with focus on an optimization of the LA-ICP-MS parameters such as laser beam diameter, laser beam fluence, and scan speed using CdS quantum dots (QDs) as a template and dopamine as a functional monomer. A non-covalent imprinting approach was employed in this study due to the simplicity of preparation. Simple oxidative polymerization of the dopamine that creates the self-assembly monolayer seems to be an ideal choice. The QDs prepared by UV light irradiation synthesis were stabilized by using mercaptosuccinic acid. Formation of a complex of QD-antibody and QD-antibody-antigen was verified by using capillary electrophoresis with laser-induced fluorescence detection. QDs and antibody were connected together via an affinity peptide linker. LA-ICP-MS was employed as a proof-of-concept for detection method of two types of immunoassay: 1) antigen extracted from the sample by MIP and subsequently overlaid/immunoreacted by QD-labelled antibodies, 2) complex of antigen, antibody, and QD formed in the sample and subsequently extracted by MIP. The first approach provided higher sensitivity (MIP/NIP), however, the second demonstrated higher selectivity. A mixture of proteins with size in range 10-250 kDa was used as a model sample to demonstrate the capability of both approaches for detection of IgG in a complex sample.
- MeSH
- elektroforéza kapilární MeSH
- fluorescence MeSH
- hmotnostní spektrometrie * MeSH
- imunoanalýza metody MeSH
- imunoglobulin G analýza MeSH
- kvantové tečky chemie MeSH
- laserová terapie * MeSH
- molekulový imprinting * MeSH
- myši MeSH
- počítačové zpracování signálu MeSH
- polymery chemie MeSH
- proteiny analýza MeSH
- sloučeniny kadmia chemie MeSH
- sulfidy chemie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The microRNA (miRNA) belongs to short noncoding RNA molecules (generally 20-25 nt in length) which are able of specific gene regulation expression 1. These regulatory molecules have significant influence on different biological processes like cell proliferation, development, differentiation, apoptosis and metabolism 2. The aberrant expression of various miRNAs is involved in the cell pathological status formation. The abnormal miRNA expression levels were found at number diseases like inflammatory and autoimmune diseases, diabetes, cardiovascular and neurodegenerative diseases and cancer 3-6. Specific miRNAs expression profiles were identified at various tumors so miRNA are interesting diagnostic biomarkers. Moreover their detection might be helpful to find more about cancer staging, prognosis and/or response to treatment 7. Different studies have shown that miR-150 is upregulated in lung cancer tissue and lupus nephritis, downregulated at patients with heart failure, acute myeloblastic leukemia, colorectal cancer and systemic sclerosis 8-14. Because of miRNAs importance the simple and fast detection method is needed. Therefore, the sensitive electrochemical analysis was combined with specific magnetic particles-based isolation.
Dot-blot is one of biological methods that are normally used in research laboratories and especially in the diagnostics. It is the most commonly used method for identification and immunodetection of particular proteins which may be markers of various diseases. The main aim of the experiment was to develop a simple, inexpensive and rapid method for specific detection of nucleic acids, especially DNA, and then this procedure apply to the detection of DNA modified by platinum cytostatic drugs. Despite platinum cytostatic drugs‘ common use in chemotherapy of various cancer types, their biochemical effect is still not completely clear. The generally accepted opinion is that the drug binds to cellular DNA. It was observed that cisplatin is bound to the DNA the most compared to oxaliplatin and carboplatin.
- Klíčová slova
- oxaliplatina,
- MeSH
- cisplatina MeSH
- DNA * imunologie účinky léků MeSH
- imunoblotting * metody statistika a číselné údaje MeSH
- karboplatina MeSH
- organoplatinové sloučeniny MeSH
- polymerázová řetězová reakce MeSH
- protilátky MeSH
- protinádorové látky * MeSH
- Publikační typ
- hodnotící studie MeSH
- práce podpořená grantem MeSH
