A single-step, highly specific and easy-to-use method was developed for isolation and purification of melatonin from complex biological matrices. Polyclonal antibodies highly specific against melatonin (with cross-reactivities with related compounds below 0.02%, except for 6-hydroxymelatonin) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of immunoaffinity gel. Melatonin recovery by the immunoaffinity method was approximately 95%, allowing single-step processing of samples prior to electrospray HPLC-MS analysis (with detection limit 10 fmol). The method was successfully used for determining melatonin in human serum and turned out to be better than the non-specific solid-phase extraction published earlier.

• MeSH
• chromatografie afinitní metody   MeSH
• ELISA MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• melatonin izolace a purifikace   MeSH
• senzitivita a specificita MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zkřížené reakce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• melatonin MeSH

Polyclonal antibodies with high specificity for C1-immobilised (+)-cis,trans-abscisic acid (ABA) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of an immunoaffinity chromatography (IAC) gel. The detection limit of the ELISA was approximately 4.6x10(-10)mol/L. Sensitive electrospray liquid chromatography-mass spectrometry (LC-ESI-MS) methods were also developed with detection limits below 0.1x10(-12)mol. The IAC allowed quick, single-step processing of samples prior to the analyses. The LC-ESI-MS and LC-ELISA techniques were used for comparative estimation of endogenous ABA levels in immunoaffinity purified extracts of normal and water-stressed Nicotiana tabacum L. leaves. The analytical approaches were validated using deuterium- and tritium-labelled internal standards, respectively. The IAC method was found to be highly effective, sensitive and convenient for isolating the target analyte from plant material.

• MeSH
• chromatografie afinitní metody   MeSH
• chromatografie kapalinová metody   MeSH
• ELISA metody   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• kyselina abscisová analýza   chemie   imunologie   MeSH
• listy rostlin chemie   účinky léků   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• tabák chemie   účinky léků   metabolismus   MeSH
• voda metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina abscisová MeSH
• voda MeSH

Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

• Klíčová slova
• Brassica napus, Brassinosteroids, Enzyme immunoassay, Immunoaffinity chromatography, Liquid chromatography-tandem mass spectrometry, Monoclonal antibodies,
• MeSH
• Brassica napus chemie   MeSH
• brassinosteroidy analýza   izolace a purifikace   MeSH
• chromatografie afinitní metody   MeSH
• imobilizační protilátky chemie   MeSH
• imunosorbenty chemie   MeSH
• regulátory růstu rostlin analýza   izolace a purifikace   MeSH
• rostlinné extrakty chemie   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• brassinosteroidy MeSH
• imobilizační protilátky MeSH
• imunosorbenty MeSH
• regulátory růstu rostlin MeSH
• rostlinné extrakty MeSH

Whey, a by-product of cheese production, is a potential source of proteins. Immunization of dairy cows in mid-lactation with mouse IgG and dinitrophenyl-keyhole limpet hemocyanin resulted in the formation of antibodies to these antigens in both blood serum and milk. The antibodies remained in whey during cheese making, and were isolated by immunoaffinity chromatography on matrices with immobilized antigens. The isolated monospecific antibodies were pure and retained their reactivity to antigens.

• MeSH
• chromatografie afinitní metody   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• ELISA MeSH
• hemokyanin MeSH
• imunoglobulin G MeSH
• mléčné bílkoviny imunologie   MeSH
• monoklonální protilátky MeSH
• myši MeSH
• skot MeSH
• syrovátkové proteiny MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemokyanin MeSH
• imunoglobulin G MeSH
• keyhole-limpet hemocyanin MeSH Prohlížeč
• mléčné bílkoviny MeSH
• monoklonální protilátky MeSH
• syrovátkové proteiny MeSH

Immunosorbents for the plant hormones cytokinins prepared by random antibody immobilization (to Affi-Gel 10) and by oriented approach via oxidized carbohydrate moieties on the Fc region (to Affi-Gel Hz or hydrazide derivative of Perloza MT 200) have been compared. Both approaches yielded immunosorbents with high dynamic capacity (ca. 5-10 nmol ml gel-1). Oriented antibody immobilization did not exhibit crucial effects in the case of low-molecular-mass cytokinins. Antibodies immobilized via a spacer to Affi-Gel 10 have probably enough conformational freedom to enable good accessibility to cytokinins. The sorbents were used in analysis of endogenous cytokinins in maize seeds. In phosphatase treated samples trans-zeatin and its riboside were predominant.

• MeSH
• celulosa MeSH
• chromatografie afinitní MeSH
• cytokininy chemie   MeSH
• imunochemie MeSH
• imunoglobulin G chemie   MeSH
• molekulární konformace MeSH
• oxidace-redukce MeSH
• protilátky chemie   MeSH
• semena rostlinná chemie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• celulosa MeSH
• cytokininy MeSH
• imunoglobulin G MeSH
• protilátky MeSH

The use of contaminated raw materials can lead to the transfer of mycotoxins into the final product, including beer. This study describes the use of the commercially available immunoaffinity column 11+Myco MS-PREP® and UPLC-MS/MS for the determination of mycotoxins in pale lager-type beers brewed in Czech Republic and other European countries. The additional aim of the work was to develop, optimize and validate this analytical method. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy were tested. The calibration curves were linear with correlation coefficients (R2 > 0.99) for all mycotoxins under investigation. The LOD ranged from 0.1 to 50 ng/L and LOQ from 0.4 to 167 ng/L. Recoveries of the selected analytes ranged from 72.2 to 101.1%, and the relative standard deviation under conditions repeatability (RSDr) did not exceed 16.3% for any mycotoxin. The validated procedure was successfully applied for the analysis of mycotoxins in a total of 89 beers from the retail network. The results were also processed using advanced chemometric techniques and compared with similar published studies. The toxicological impact was taken into account.

• Klíčová slova
• 11+Myco MS-PREP®, Beer, Dietary exposure, Immunoaffinity columns, Mycotoxins, UPLC-MS/MS,
• MeSH
• chromatografie kapalinová metody   MeSH
• dietární expozice analýza   MeSH
• mykotoxiny * analýza   MeSH
• pivo analýza   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mykotoxiny * MeSH

A method for the immobilization of antibodies to inert matrix represents an important factor that affects results of immunoaffinity chromatography. Binding antibodies to immobilized metal ions is an example of oriented immobilization that avoids a random coupling of a protein. Preparation of a stable immunoaffinity sorbent using immobilized metal ions was described. Antibodies were bound to chelated Co3+ ions that were prepared by oxidation of Co2+-iminodiacetic acid agarose using hydrogen peroxide. The formation of a stable complex of the antibody with immobilized Co3+ ions was proved. Antibodies bound by this way were not released with solutions of 50 mM EDTA, 6 M urea, 3 M NaCl, 20% v/v dioxane, 0.1 M imidazole and buffers of pH 2.5 and pH 11.0. If needed, antibody could be released from the carrier by the reduction of Co3+ ions with a reducing agent (e.g. dithiotreitol or 2-mercaptoethanol). Antibody released from the carrier could be then replaced by another antibody. The method described in this paper was used for the immobilization of polyclonal rabbit anti-ovalbumin antibody or egg yolk antibody (IgY) produced in chicken. In a model experiment, immobilized polyclonal rabbit antibodies were used for the separation of ovalbumin from egg white and conditions of chromatography were described.

• MeSH
• chromatografie afinitní metody   MeSH
• imunoglobulin G chemie   metabolismus   MeSH
• imunoglobuliny chemie   metabolismus   MeSH
• kobalt chemie   MeSH
• králíci MeSH
• kur domácí MeSH
• oxidancia chemie   MeSH
• peroxid vodíku chemie   MeSH
• protilátky chemie   metabolismus   MeSH
• sefarosa chemie   MeSH
• vaječné proteiny chemie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• IgY MeSH Prohlížeč
• imunoglobulin G MeSH
• imunoglobuliny MeSH
• kobalt MeSH
• oxidancia MeSH
• peroxid vodíku MeSH
• protilátky MeSH
• sefarosa MeSH
• vaječné proteiny 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
• chromatografie afinitní MeSH
• fenotyp MeSH
• haptoglobiny analýza   imunologie   MeSH
• lidé MeSH
• povrchové vlastnosti MeSH
• protilátky imunologie   MeSH
• software MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• haptoglobiny MeSH
• protilátky MeSH

An analytical protocol for the isolation and quantification of indole-3-acetic acid (IAA) and its amino acid conjugates was developed. IAA is an important phytohormone and formation of its conjugates plays a crucial role in regulating IAA levels in plants. The developed protocol combines a highly specific immunoaffinity extraction with a sensitive and selective LC-MS/MS analysis. By using internal standards for each of the studied compounds, IAA and seven amino acid conjugates were analyzed in quantities of fresh plant material as low as 30 mg. In seeds of Helleborus niger, physiological levels of these compounds were found to range from 7.5 nmol g(-1) fresh weight (IAA) to 0.44 pmol g(-1) fresh weight (conjugate with Ala). To our knowledge, the identification of IAA conjugates with Gly, Phe and Val from higher plants is reported here for the first time.

• MeSH
• chromatografie afinitní přístrojové vybavení   metody   MeSH
• fenylalanin chemie   MeSH
• glycin chemie   MeSH
• Helleborus chemie   MeSH
• kyseliny indoloctové chemie   imunologie   izolace a purifikace   MeSH
• monoklonální protilátky imunologie   MeSH
• referenční standardy MeSH
• regulátory růstu rostlin imunologie   izolace a purifikace   normy   MeSH
• semena rostlinná chemie   MeSH
• specificita protilátek MeSH
• spektrofotometrie ultrafialová MeSH
• tandemová hmotnostní spektrometrie MeSH
• valin chemie   MeSH
• vysokoúčinná kapalinová chromatografie přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fenylalanin MeSH
• glycin MeSH
• indoleacetic acid MeSH Prohlížeč
• kyseliny indoloctové MeSH
• monoklonální protilátky MeSH
• regulátory růstu rostlin MeSH
• valin MeSH

Milk thistle [Silybum marianum (L.) Gaertn.] achieved a significant increase in interest over the past few years from local and foreign pharmaceutical corporations. The silymarin complex of constituents extracted from milk thistle achenes provides compelling health benefits primarily thanks to antioxidant activities and hepatoprotective effects. However, consuming mycotoxin-contaminated plant material can cause immunosuppression and hepatotoxic problems. The aim of this study was to develop and validate a method for the determination of mycotoxin content in milk thistle. Fusarium toxins as T-2 and HT-2 toxins in grown milk thistle harvested from a breeding station in the Czech Republic during 2020-2021 were studied. The analysis of T-2 and HT-2 toxins was performed by UPLC-MS/MS after immunoaffinity columns EASI-EXTRACT® T-2 & HT-2 clean up. All analysed samples of milk thistle were contaminated with T-2 toxin and HT-2 toxin. The content of T-2 toxin in the samples from 2020 was in the range of 122.7-290.2 µg/kg and HT-2 toxin 157.0-319.0 µg/kg. In 2021, the content of T-2 toxin was in the range of 28.8-69.9 µg/kg and HT-2 toxin was 24.2-75.4 µg/kg. The results show that the climatic conditions of the year of harvesting have a highly statistically significant effect on the content of T-2 and HT-2 toxins in milk thistle.

• Klíčová slova
• HT-2 toxin, T-2 toxin, UPLC-MS/MS, immunoaffinity column, milk thistle, validation method,
• MeSH
• antioxidancia MeSH
• biologické přípravky * MeSH
• chromatografie kapalinová MeSH
• flavonoidy MeSH
• mykotoxiny * MeSH
• ostropestřec mariánský MeSH
• semena rostlinná MeSH
• silymarin * MeSH
• šlechtění rostlin MeSH
• T-2 toxin * analogy a deriváty   MeSH
• tandemová hmotnostní spektrometrie MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antioxidancia MeSH
• biologické přípravky * MeSH
• flavonoidy MeSH
• HT-2 toxin MeSH Prohlížeč
• mykotoxiny * MeSH
• silymarin * MeSH
• T-2 toxin * MeSH