Melanoma is a skin cancer with permanently increasing incidence and resistance to therapies in advanced stages. Reports of spontaneous regression and tumour infiltration with T-lymphocytes makes melanoma candidate for immunotherapies. Cytokines are key factors regulating immune response and intercellular communication in tumour microenvironment. Cytokines may be used in therapy of melanoma to modulate immune response. Cytokines also possess diagnostic and prognostic potential and cytokine production may reflect effects of immunotherapies. The purpose of this review is to give an overview of recent advances in proteomic techniques for the detection and quantification of cytokines in melanoma research. Approaches covered span from mass spectrometry to immunoassays for single molecule detection (ELISA, western blot), multiplex assays (chemiluminescent, bead-based (Luminex) and planar antibody arrays), ultrasensitive techniques (Singulex, Simoa, immuno-PCR, proximity ligation/extension assay, immunomagnetic reduction assay), to analyses of single cells producing cytokines (ELISpot, flow cytometry, mass cytometry and emerging techniques for single cell secretomics). Although this review is focused mainly on cancer and particularly melanoma, the discussed techniques are in general applicable to broad research field of biology and medicine, including stem cells, development, aging, immunology and intercellular communication.

• MeSH
• Protein Array Analysis MeSH
• Cytokines analysis   MeSH
• Mass Spectrometry MeSH
• Immunoassay MeSH
• Immunotherapy MeSH
• Humans MeSH
• Melanoma diagnosis   metabolism   therapy   MeSH
• Tumor Microenvironment MeSH
• Skin Neoplasms diagnosis   metabolism   therapy   MeSH
• Proteomics MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners).

• MeSH
• Cell Culture Techniques MeSH
• Protein Array Analysis methods   MeSH
• Cytokines metabolism   MeSH
• Immunoassay methods   MeSH
• Stem Cells physiology   MeSH
• Culture Media, Conditioned metabolism   MeSH
• Cells, Cultured MeSH
• Cell Communication * MeSH
• Intercellular Signaling Peptides and Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Edukační publikace se zabývá v první části využitím čipů v imunochemii pro mikroanalýzu. Je uveden přehled podkladových materiálů a značek, jakož i senzitivita jednotlivých technologií. Vysvětluje se možnost imobilizace a detekce proteinů pomocí planárního vlnovodu. Ukazuje se používání čipů při paralelní kompetitivní imunoanalýze, sendvičové imunoanalýze a fluorescenční imunoanalýze na mikrokuličkách. Druhá část je věnována kapilární elektroforéze a jejímu využití k separaci po kompetitivní a nekompetitivní homogenní imunoanalýze, a heterogenní imunoanalýze. Diskutují se možnosti detekce analytu jako laserem indukovaná fluorescence, enzymové značky, chemiluminiscence, ampérometrie, UV/VIS absorbance, hmotnostní spektrometrie a povrchová plazmonová rezonance.

The first part of the educational article covers chip application for microarray in immunochemistry. An outline of surface chemistries and labelings is presented, as well as the sensitivity of various technologies. Protein immobilization and detection using planar waveguide technology is clarified. Chip applications are shown in parallel competitive immunoassay, sandwich immunoassay and fluorescent microsphere immunoassay. The second part covers capillary electrophoresis and its use for separation after competitive and non-competitive homogeneous immunoassay or heterogeneous immunoassay. The following possibilities of analyte detection are discussed: laser-induced fluorescence, enzyme labels, chemiluminiscence, amperometry, UV/VIS absorbance, mass spectrometry and surface plasmon resonance.

• MeSH
• Protein Array Analysis MeSH
• Electrophoresis, Capillary methods   MeSH
• Spectrometry, Fluorescence methods   MeSH
• Mass Spectrometry MeSH
• Immunoassay * methods   MeSH
• Luminescence MeSH
• Microarray Analysis methods   MeSH
• Surface Plasmon Resonance MeSH
• Sensitivity and Specificity MeSH

BACKGROUND: Recent studies show that the haptoglobin phenotype in individuals with diabetes mellitus is an important factor for predicting the risk of myocardial infarction, cardiovascular death, and stroke. Current methods for haptoglobin phenotyping include PCR and gel electrophoresis. A need exists for a reliable method for high-throughput clinical applications. Mass spectrometry (MS) can in principle provide fast phenotyping because haptoglobin α 1 and α 2, which define the phenotype, have different molecular masses. Because of the complexity of the serum matrix, an efficient and fast enrichment technique is necessary for an MS-based assay. METHODS: MALDI plates were functionalized by ambient ion landing of electrosprayed antihaptoglobin antibody. The array was deposited on standard indium tin oxide slides. Fast immunoaffinity enrichment was performed in situ on the plate, which was further analyzed by MALDI-TOF MS. The haptoglobin phenotype was determined from the spectra by embedded software script. RESULTS: The MALDI mass spectra showed ion signals of haptoglobin α subunits at m/z 9192 and at m/z 15 945. A cohort of 116 sera was analyzed and the reliability of the method was confirmed by analyzing the identical samples by Western blot. One hundred percent overlap of results between the direct immunoaffinity desorption/ionization MS and Western Blot analysis was found. CONCLUSIONS: MALDI plates modified by antihaptoglobin antibody using ambient ion landing achieve low nonspecific interactions and efficient MALDI ionization and are usable for quick haptoglobin phenotyping.

• MeSH
• Chromatography, Affinity MeSH
• Phenotype MeSH
• Haptoglobins analysis   immunology   MeSH
• Humans MeSH
• Surface Properties MeSH
• Antibodies immunology   MeSH
• Software MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Personalized medicine is partly based on biomarker-guided diagnostics, therapy and prognosis, which is becoming an unavoidable concept in modern cardiology. However, the clinical significance of single biomarker studies is rather limited. A promising novel approach involves combining multiple markers into a multiplex panel, which could refine the management of a particular patient with cardiovascular pathology. Two principally different assay formats have been developed to facilitate simultaneous quantification of multiple antigens: planar array assays and microbead assays. These approaches may help to better evaluate the complexity and dynamic nature of pathologic processes and offer substantial cost and sample savings compared with traditional enzyme-linked immunosorbent assay (ELISA) measurements. However, a multiplex multimarker approach cannot become a generally disseminated method until analytical problems are solved and further studies confirming improved clinical outcomes are accomplished. These drawbacks underlie the fact that a limited number of systematic studies are available regarding the use of a multiplex biomarker approach in cardiovascular medicine to date. Our perspective underscores the significant potential of the use of the multiplex approach in a wider conceptual framework under the close cooperation of clinical and experimental cardiologists, pathophysiologists and biochemists so that the personalized approach based on standardized multimarker testing may improve the management of various cardiovascular pathologies and become a ubiquitous partner of population-derived evidence-based medicine.

• MeSH
• Biomarkers analysis   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Cardiovascular Diseases diagnosis   drug therapy   MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Multiplexní analýza umožňuje současné stanovení více analytů v tomtéž vzorku. Tyto postupy, původně rozšířené především v genomice, nyní významně pronikají i do dalších oblastí bioanalytiky. V analýze proteinů hrají zásadní roli imunochemické metody a jejich multiplexace a miniaturizace je široce využitelná jak v základním, tak v aplikovaném výzkumu. Také možnosti uplatnění těchto vysokokapacitních metod v laboratorní diagnostice jsou značné. Text popisuje dva hlavní přístupy k multiplexní imunoanalýze – provedení v planárním a suspenzním uspořádání. Krátce diskutuje principy, detekční metody a přednosti obou těchto strategií. V závěru jsou zmíněna některá úskalí spojená s integrací multiplexní analýzy do diagnostiky.

Multiplex analysis enables a simultaneous determination of multiple targets in one sample. This approach has been largely adopted in genomics and progressively expands to various domains of bioanalytics. In protein analysis, immunoassays play a fundamental role and their multiplexing and miniaturization is of great applicability to both basic and applied research. Furthermore, these high-throughput methodologies have a considerable potential in the field of laboratory diagnostics. The following text describes planar and bead-based arrays, two main strategies of immunoassay multiplexing. Principles, detection methods and strengths of each are shortly discussed. Finally, we mention several challenges linked with the integration of these methods to diagnostics.

• Keywords
• multiplexní analýza,
• MeSH
• Biosensing Techniques methods   instrumentation   MeSH
• Financing, Organized MeSH
• Immunoassay methods   MeSH
• Microarray Analysis methods   instrumentation   MeSH
• Flow Cytometry methods   instrumentation   MeSH

• MeSH
• Cell Biology MeSH
• Molecular Biology MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• biologie
• cytologie, klinická cytologie