Due to their unique three-dimensional structure, DNA or RNA oligonucleotide aptamers bind to various molecules with high affinity and specificity. Aptamers, alone or in combination with antibodies, can be used to sensitively quantify target molecules by quantitative real-time polymerase chain reaction (qPCR). However, the assays are often complicated and unreliable. In this study, we explored the feasibility of performing the entire assay on wells of routinely used polypropylene PCR plates. We found that polypropylene wells efficiently bind proteins. This allows the entire assay to be run in a single well. To minimize nonspecific binding of the assay components to the polypropylene wells, we tested various blocking agents and identified methylcellulose as an effective alternative to the commonly used BSA. Methylcellulose not only demonstrates comparable or superior blocking capabilities but also offers the advantage of a well-defined composition and non-animal origin. Our findings support the utilization of aptamers, either alone or in combination with antibodies, for sensitive quantification of selected molecules immobilized in polypropylene PCR wells in a streamlined one-well qPCR assay under well-defined conditions.

• Klíčová slova
• DNA aptamers, blocking agents, immunoglobulins, methylcellulose, polymerase chain reaction, polypropylene wells,
• MeSH
• aptamery nukleotidové * MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• methylcelulosa MeSH
• polypropyleny MeSH
• protilátky MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aptamery nukleotidové * MeSH
• methylcelulosa MeSH
• polypropyleny MeSH
• protilátky MeSH

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.

• Klíčová slova
• T-cell, biomarker, cancer, cytokine, immunoassay, mass spectrometry, melanoma, proteomics, secretome, ultrasensitive,
• MeSH
• čipová analýza proteinů MeSH
• cytokiny analýza   MeSH
• hmotnostní spektrometrie MeSH
• imunoanalýza MeSH
• imunoterapie MeSH
• lidé MeSH
• melanom diagnóza   metabolismus   terapie   MeSH
• nádorové mikroprostředí MeSH
• nádory kůže diagnóza   metabolismus   terapie   MeSH
• proteomika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• cytokiny MeSH