ELISA-like Assay Research Articles

NEW MOLECULAR RECEPTORS FOR DIAGNOSIS AND THERAPY OF PANCREATIC DUCTAL ADENOCARCINOMA

Abstract INTRODUCTION Pancreatic ductal adenocarcinoma (PDAC) is one of the cancers with worst prognosis due to the intense desmoplastic reaction that occurs in this disease, which hinders the action of therapeutic agents and contributes to cancer progression. A possible way to develop new strategies for PDAC diagnosis and therapy is the study of the abnormal components of the tumor stroma. However, new receptors capable of detecting these components are needed. MATERIALS AND METHODS Two types of molecular receptors, monoclonal antibodies and aptamers, were developed in parallel targeting an extracellular matrix protein with potential as a PDAC marker. Antibodies were generated using mice of the DBA/1J strain, while aptamers were obtained by means of an in vitro selection method called SELEX. The analytical characteristics of the developed receptors were studied by ELISA-like assays, as well as cytochemistry on positive and negative cell lines for the protein of interest. RESULTS A total of 44 potential aptamers were identified. After a first bioinformatic screening, 9 candidates were chosen for evaluating their binding affinity in vitro. Two of them showed dissociation constants in the nanomolar range. One of the aptamers also showed specific staining of cells positive for the marker protein, as did one of the generated antibody clones. CONCLUSIONS The newly developed receptors have shown a great potential in the study of PDAC. However, their diagnostic and therapeutic usefulness requires a more in-depth evaluation, currently in progress. ACKNOWLEDGEMENTS This research was funded by the Spanish Government (RTI-2018-095756-B-I00) and Principado de Asturias Government (IDI2018-000217), co-financed by FEDER funds. R.L.G. thanks the Spanish Government for a predoctoral FPU fellowship (FPU16/05670).

Sr-Containing Mesoporous Bioactive Glasses Bio-Functionalized with Recombinant ICOS-Fc: An In Vitro Study.

Osteoporotic bone fractures represent a critical clinical issue and require personalized and specific treatments in order to stimulate compromised bone tissue regeneration. In this clinical context, the development of smart nano-biomaterials able to synergistically combine chemical and biological cues to exert specific therapeutic effects (i.e., pro-osteogenic, anti-clastogenic) can allow the design of effective medical solutions. With this aim, in this work, strontium-containing mesoporous bioactive glasses (MBGs) were bio-functionalized with ICOS-Fc, a molecule able to reversibly inhibit osteoclast activity by binding the respective ligand (ICOS-L) and to induce a decrease of bone resorption activity. N2 adsorption analysis and FT-IR spectroscopy were used to assess the successful grafting of ICOS-Fc on the surface of Sr-containing MBGs, which were also proved to retain the peculiar ability to release osteogenic strontium ions and an excellent bioactivity after functionalization. An ELISA-like assay allowed to confirm that grafted ICOS-Fc molecules were able to bind ICOS-L (the ICOS binding ligand) and to investigate the stability of the amide binding to hydrolysis in aqueous environment up to 21 days. In analogy to the free form of the molecule, the inhibitory effect of grafted ICOS-Fc on cell migratory activity was demonstrated by using ICOSL positive cell lines and the ability to inhibit osteoclast differentiation and function was confirmed by monitoring the differentiation of monocyte-derived osteoclasts (MDOCs), which revealed a strong inhibitory effect, also proven by the downregulation of osteoclast differentiation genes. The obtained results showed that the combination of ICOS-Fc with the intrinsic properties of Sr-containing MBGs represents a very promising approach to design personalized solutions for patients affected by compromised bone remodeling (i.e., osteoporosis fractures).

Latent 1,3-β-D-glucan acts as an adjuvant for allergen-specific IgE production induced by Japanese cedar pollen exposure

BackgroundThe pollen grains of several plant species contain 1,3-β-D-glucan (BG). BG activates dendritic cells (DCs) and subsequently regulates the innate immune responses. Within Japan, the most common disease associated with type-I hypersensitivity is Japanese cedar pollinosis. However, the role of BG in Japanese cedar pollen (JCP) remains unclear. This study examined the localization and immunological effects of BG in JCP. MethodsThe localization of BG in JCP grain was determined by immunohistochemical staining using a soluble dectin-1 protein probe and a BG recognition protein (BGRP). The content of BG extracted from JCP was measured by a BGRP-based ELISA-like assay. The cytokine production by bone marrow-derived DCs (BMDCs) obtained from wild-type and BG receptor (dectin-1) knock-out mice was examined in vitro. The mice were intranasally administered JCP grains and the specific serum Ig levels were then quantified. ResultsBG was detected in the exine and cell wall of the generative cell and tube cell of the JCP grain. Moreover, BG in the exine stimulated production of TNF-α and IL-6 in the BMDCs via a dectin-1-dependent mechanism. Meanwhile, JCP-specific IgE and IgG were detected in the serum of wild-type mice that had been intranasally administered with JCP grains. These mice also exhibited significantly enhanced sneezing behavior. However, dectin-1 knock-out mice exhibited significantly lower JCP-specific IgE and IgG levels compared to wild-type mice. ConclusionsLatent BG in JCP can act as an adjuvant to induce JCP-specific antibody production via dectin-1.

Antiviral and virucidal activities of sulphated polysaccharides against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), a member of the family Flaviviridae, causes severe neurological disorders in humans. JEV infections represent one of the most widely spread mosquito-borne diseases, and therefore, it has been considered as an endemic disease. An effective antiviral drug is still unavailable to treat JEV, and current drugs only provide supportive treatment to alleviate the symptoms and stabilize patients' conditions. This study was designed to evaluate the antiviral activity of the sulphated polysaccharides "Carrageenan," a linear sulphated polysaccharide that is extracted from red edible seaweeds against JEV replication in vitro. Viral inactivation, attachment, and post-infection assays were used to determine the mode of inhibition of Carrageenan. Virus titters after each application were evaluated by plaque formation assay. MTT assay was used to determine the 50% cytotoxic concentration (CC50), and ELISA-like cell-based assay and immunostaining and immunostaining techniques were used to evaluate the 50% effective concentration (EC50). This study showed that Carrageenan inhibited JEV at an EC50 of 15 µg/mL in a dose-dependent manner with CC50 more than 200 µg/mL in healthy human liver cells (WRL68). The mode of inhibition assay showed that the antiviral effects of Carrageenan are mainly due to their ability to inhibit the early stages of virus infection such as the viral attachment and the cellular entry stages. Our investigation showed that Carrageenan could be considered as a potent antiviral agent to JEV infection. Further experimental and clinical studies are needed to investigate the potential applications of Carrageenan for clinical intervention against JEV infection.

Advanced ELISA-like Biosensing Based on Ultralarge-Pore Silica Microbeads.

In this work, functionalized porous silica-based materials, widely used in the literature as drug and biomolecule nanocarriers, were innovatively used as an effective three-dimensional (3D) substrate for the development of a specific biomolecular assay showing great versatility in terms of detection performance. One-pot synthesis of ultralarge-pore silica microbeads was optimized to develop an enzyme-linked immunosorbent (ELISA)-like DNA detection assay. Cocondensation synthesis enabled introducing thiol functionalities into the silica framework while preserving both the high specific surface area (560 m2/g) and large pore size (17 nm average diameter), which are essential to guaranteeing high loading capability. Indeed, the bead-capturing ability was proved by developing an ELISA-like assay for the detection of short DNA sequences (≈20 bp), both in labeled and label-free configurations. In particular, the suppression of unspecific binding on the bead surface by testing two different blocking agents was a matter of interest. The detection performances were evaluated and compared to the ones obtained by following the same detection protocol on a standard flat surface (two-dimensional, 2D), which is most commonly used for this purpose. The bead-based assay showed a limit of detection two times lower than the flat-surface assay, confirming the promising capturing ability due to the larger active surface area. Furthermore, compared to traditional ELISA, the bead-based assay showed an intrinsic larger dynamic range that can be tailored depending on the final amount of beads used for the colorimetric quantification.

Localization of Therapeutic Fab-CHP Conjugates to Sites of Denatured Collagen for the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in synovial joints and protease-induced cartilage degradation. Current biologic treatments for RA can effectively reduce symptoms, primarily by neutralizing the proinflammatory cytokine TNFα; however, continued, indiscriminate overinhibition of inflammatory factors can significantly weaken the host immune system, leading to opportunistic infections and interrupting treatment. We hypothesize that localizing anti-TNFα therapeutics to denatured collagen (dCol) present at arthritic joints, via conjugation with collagen-hybridizing peptides (CHPs), will reduce off-site antigen binding and maintain local immunosuppression. We isolated the antigen-binding fragment of the clinically approved anti-TNFα therapeutic infliximab (iFab) and prepared iFab-CHP conjugates via lysine-based conjugation with an SMCC linker. After successful conjugation, confirmed by LC-MS, the binding affinity of iFab-CHP was characterized by ELISA-like assays, which showed comparable antigen binding relative to infliximab, comparable dCol binding relative to CHP, and the hybrid ability to bind both dCol and TNFα simultaneously. We further demonstrated localization of Fab-CHP to areas of high dCol in vivo and promising therapeutic efficacy, assessed by histological staining (Safranin-O and H&E), in a pilot mouse study.

Abstract A14: Detection of a biomarker for B-cell non-Hodgkin lymphomas or leukemias using circulating tumor DNA without the use of PCR or next-gen sequencing

Abstract There is a critical need for the development of cheap, safe, and accurate tools for cancer surveillance. Patients diagnosed with cancer require an accurate assessment of tumor burden through the course of their treatment, and the need for tumor surveillance is even more acute for many indolent B-cell malignancies such as follicular lymphoma and chronic lymphocytic leukemia. Over the past several years, we have developed an assay that quantifies a tumor biomarker for many different types of B-cell non-Hodgkin lymphomas or leukemias (B-NHLs). This biomarker, uracils in DNA, originates from the activity of a B-cell maturation enzyme, activation-induced deaminase (AID), which converts cytosines in DNA to uracil. AID is normally not expressed in circulating B cells or any other human tissue and hence this marker is not present in any other types of cells in the human body. We have developed an ELISA-like assay using chemicals with a fluorescence readout that can detect this biomarker in DNA with high sensitivity without PCR or NGS. We will present applications of this technology to genomic DNA from B-NHL tumors and circulating tumor DNA in blood plasma of such patients. Our data show we can detect the presence of this marker in DNA of patients with follicular lymphoma, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia. Citation Format: Jessica A. Stewart, Ashok S. Bhagwat. Detection of a biomarker for B-cell non-Hodgkin lymphomas or leukemias using circulating tumor DNA without the use of PCR or next-gen sequencing [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A14.

A sandwich ELISA-like detection of C-reactive protein in blood by citicoline-bovine serum albumin conjugate and aptamer-functionalized gold nanoparticles nanozyme

C-reactive protein (CRP) level in blood is associated with the risk of developing cardiovascular events in higher-risk populations. We present a sandwich ELISA-like assay for the determination of CRP in blood by citicoline-bovine serum albumin (citicoline-BSA) conjugate and aptamer-functionalized gold nanoparticles (aptamer-AuNPs) nanozyme. The CRP in the blood sample was selectively adsorbed to the ELISA plate coated by citicoline-BSA, and then incubated with added aptamer-AuNPs. AuNPs exhibited peroxidase activity and oxidized 3,3′5,5′-tetramethylbenzidine from colorless to blue, achieving the measurement at 652 nm. The amplified signal increased linearly in a wide range from 0.1 to 200 ng mL−1 and with a detection limit of 8 pg mL−1. Finally, the method was further tested using rat blood from an isoproterenol-induced myocardial infarction experimental model to confirm its applicability. The developed method could directly determine CRP in blood sample after dilution with high accuracy and sensitivity. This method has many advantages, such as easiness to prepare materials, good stability between batches, high specificity, low detection limit, low-cost, easiness to operate with simple instruments, the most remarkable of which is its excellent lot-to-lot stability over the classical ELISA.

β2-adrenergic stimulation induces interleukin-6 by increasing Arid5a, a stabilizer of mRNA, through cAMP/PKA/CREB pathway in cardiac fibroblasts.

Background and PurposeIn cardiovascular diseases, cardiac fibroblasts (CFs) participate in the myocardial inflammation by producing pro‐inflammatory cytokines, worsening the prognosis. β2‐adrenergic receptor (AR) and β3AR are expressed in CFs, and β‐adrenergic stimulation promotes CFs to produce pro‐inflammatory cytokines. However, the mechanism of the expression of pro‐inflammatory cytokines in response to β‐adrenergic stimulation remains to be fully elucidated.Experimental ApproachCFs were isolated from adult wild‐type or AT‐rich interactive domain‐containing protein 5A (Arid5a) knockout mice. The expression of mRNA was measured by real‐time RT‐PCR. Interleukin (IL)‐6 protein was measured by ELISA. The activity of nuclear factor‐κB (NF‐κB) and cyclic AMP (cAMP) response element binding protein (CREB) was assessed by ELISA‐like assay or Western blotting.Key ResultsThe β‐adrenergic stimulation remarkably induced IL‐6 mRNA and protein through β2AR in CFs. The activation of adenylate cyclase and the enhancement of intracellular cAMP resulted in the upregulation of IL‐6 mRNA expression. The induction of IL‐6 transcript by β2AR signaling was independent of NF‐κB. Concomitant with IL‐6, the expression of Arid5a, an IL‐6 mRNA stabilizing factor, was enhanced by β2‐adrenergic stimulation and by cAMP increase. Importantly, β2AR signaling‐mediated IL‐6 induction was suppressed in Arid5a knockout CFs. Finally, β2AR stimulation phosphorylated CREB via PKA pathway, and the activation of CREB was essential for the induction of Arid5a and IL‐6 mRNA.Conclusion and Implicationsβ2‐adrenergic stimulation post‐transcriptionally upregulates the expression of IL‐6 by the induction of Arid5a through cAMP/PKA/CREB pathway in adult CFs. β2AR/Arid5a/IL‐6 axis could be a therapeutic target against cardiac inflammation.

Development of a novel β-1,6-glucan–specific detection system using functionally-modified recombinant endo-β-1,6-glucanase

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a KD of 16.4 nm. We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro. We also used this assay to measure β-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

Nanobody\u2011horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

BackgroundAntibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV.ResultsIn the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni–NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests.ConclusionsFor the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.

Antibody free ELISA-like assay for the detection of transcription factors based on double-stranded DNA thermostability.

Transcription factors (TFs) play critical roles in gene expression regulation and disease development. In this paper, we report an antibody free ELISA-like assay which could be used to analyze transcription factor NF-κB p50 with comparatively low cost and high throughput. This assay is based on the stabilization of a duplex DNA probe by binding with a transcription factor. The double-stranded DNA (dsDNA) probe immobilized on a 96-well plate was too short in length to stabilize its duplex structure at a relatively high temperature and would unwind into a single strand. In the presence of a target TF, the protein bound to a specific TF-binding site and prevented the specific dsDNA probe from being unzipped at the washing temperature, thus holding the chemiluminescence signal. This method could sensitively detect NF-κB p50 with a detection limit of 0.5 nM. The proposed strategy provides a convenient, cheap and high throughput detection method of TFs, which can potentially help the development of drug discovery and disease diagnosis.

Quantification of Mushroom-Derived Soluble β-1,6-Glucan Using the Function-Modified Recombinant β-1,6-Glucanase.

Mushroom is one of the major sources of β-glucan used in medical applications and traditional therapies. Thus, structure analysis and quantification of β-glucan content is crucial to evaluate medicinal mushrooms. Most studies concerning mushroom-derived β-glucan have been focused on β-1,3-glucans. However, recent investigations suggest that β-1,6 glucans have important roles for immunomodulating activity. Therefore, to elucidate the fine structure of various mushroom-derived β-glucans, we recently developed a novel β-1,6 glucan detection system using the function-modified recombinant β-1,6-glucanase. In this study, we performed an ELISA-like assay using modified β-1,6-glucanase and soluble dectin-1-Fc as the probes for β-1,6-glucan and β-1,3-glucan, respectively. Reactivity of ELISA to crude hot water extracts of edible mushrooms (Grifola frondosa, Agaricus bisporus, Pleurotus tuoliensis, P. eryngii, P. ostreatus, Hypsizygus marmoreus, and Lentinus edodes) was compared and L. edodes showed the strongest reactivity among them. An additional 19 different products of fresh L. edodes (shiitake mushroom) commercially available in Japan were also analyzed. This revealed limited differences in amounts of β-1,6-glucan and β-1,3-glucan in each shiitake mushroom. Furthermore, structural analysis of some purified β-glucans derived from medicinal mushrooms was performed, and their action for inducing tumor necrosis factor-α production from the murine bone marrow-derived dendritic cells was investigated. We found relation between reactivity to modified β-1,6-glucanase and its cytokine inducing activity. This assay could be useful for evaluating the strains of edible or medicinal mushrooms, which may be used as alternative medicines.

GENE-38. IDH1MUT INDUCES N6-METHYLADENOSINE (m6A) RNA HYPERMETHYLATION VIA D-2-HG IN GLIOMA

Abstract INTRODUCTION IDH1 MUT confers slower tumor growth rates and improved prognoses for glioma patients, but the precise mechanisms underlying these clinically relevant benefits remain largely unknown. IDH1MUT gliomas produce high levels of D-2-HG, a putative oncometabolite that inhibits a range of α-ketoglutarate-dependent dioxygenases. D-2-HG-mediated inhibition of TET-family proteins involved in DNA demethylation is known to be a key driver leading to the oncogenic DNA hypermethylation phenotype (known as G-CIMP) seen in IDH1MUT gliomas. Recent evidence indicates that D-2-HG also functionally inhibits FTO and AlkBH5, two dioxygenases responsible for demethylation of RNA N6-methyladenosine (m6A) sites. This study sought to determine if D-2-HG mediates an increase in m6A content in IDH1MUT gliomas. METHODS Total RNA was isolated from patient tumor samples, patient-derived gliomaspheres, human embryonic kidney cell (HEK293T), normal human astrocyte (NHA), and U87 glioma model systems expressing either IDH1MUT or IDH1WT. Relative abundance of m6A modifications was determined both quantitatively and qualitatively using colorimetric ELISA-like assays and m6A-antibody dot blots, respectively. RESULTS Quantification of m6A abundance in IDH1MUT patient tumor samples and patient-derived gliomaspheres revealed an increase in m6A content compared to IDH1WT samples. Forced expression of IDH1MUT in HEK293T, NHA, and U87 cells increased intracellular D-2-HG content and global m6A abundance in purified RNA. Additionally, D-2-HG treatment of IDH1WT cell lines increased m6A abundance, including in IDH1WT gliomaspheres. Conversely, inhibition of D-2-HG generation in IDH1MUT cell lines decreased m6A abundance. Data will also be presented suggesting that increased m6A abundance is associated with decreased cellular proliferation. CONCLUSIONS These results indicate that increased intracellular D-2-HG arising in the context of IDH1MUT mediates increases in RNA m6A methylation in glioma. The association with m6A hypermethylation and reduced growth suggests that RNA methylation provides a novel therapeutic target.

Flex-Array® – a novel multi-well vessel system for the immobilization and multi-modal testing of intact formalin-fixed paraffin-embedded (FFPE) cells or tissues

ABSTRACTFlex-Array® is a novel multi-well system that extends the key features of current high-performance microscope slides used in advanced staining techniques. The Flex-Array® system facilitates the immobilization of FFPE cell or tissue sections onto a multi-well array for the subsequent performance of single analyte (IHC) or multiplexed immunohistochemistry (MIHC). Additionally, the Flex-Array® device is compatible with fluorescent and colorimetric in situ hybridization (FISH) and adds new capabilities such as replicate analysis, quantitative ELISA-like assays and microdissection-free nucleic acid extraction. The Flex-Array® facilitates rapid, contextually rich and high-precision multi-modal analysis of FFPE cells and tissues at a significant reduction in testing, data acquisition and analysis costs.

Beacons Contribute Valuable Empirical Information to Theoretical 3-D Aptamer-Peptide Binding.

DNA aptamers were developed against five different peptides from the known binding regions of anti-Cytomegalovirus and anti-Herpes Simplex Virus-2 antibodies and the aptamers were ranked by relative affinity based on an ELISA-like (ELASA) microplate assay. The secondary structures of the top five highest affinity aptamers were studied for stem-loop commonalities and the most probable peptide binding sites. Two of these stem-loop structures were converted into beacons by addition of TYE 665 dye on the 5' end and Iowa Black quencher on the 3' end. When competed against increasing concentrations of each of the five peptides, only three of the possible ten interactions demonstrated "lights on" fluorescence beacon responses. When modeled by generation of PDB files, after passage through PATCHDOCK and YASARA, two of the aptamer beacon-peptide interactions showed no theoretical evidence of separating the G-C stem-loop region, despite clear empirical evidence of separation of the fluorophore and quencher beyond the Förster distance leading to abundant fluorescence. And in the second beacon's case, YASARA modeling suggested that the beacon was always open despite clear empirical evidence that it was not (no fluorescence response) and only opened in the presence of one of the five peptides. These results are interpreted as a demonstration that 3-dimensional docking software such as PATCHDOCK and YASARA, which are based on rigid receptor-ligand shape complementarity may not reflect the "induced-fit" interactions between aptamers and their cognate targets. Therefore, for the most complete and accurate picture of aptamer-peptide binding, several theoretical and empirical (e.g., beacon fluorescence) analysis methods may be needed.

Carbon Nanotube- and Asbestos-Induced DNA and RNA Methylation Changes in Bronchial Epithelial Cells.

Carbon nanotubes (CNTs) are nanoscale tube-shaped carbon materials used in many industrial areas. Their fiber shape has caused concerns about their toxicity given their structural similarity with asbestos. The aim here was to elucidate the effect of CNTs and asbestos exposure on global DNA and RNA methylation and the methylation of genes associated with cell cycle, inflammation, and DNA damage processes in human lung cells. Human bronchial epithelial cells (16HBE14o-) were exposed for 24 h to 25 and 100 μg/mL CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) and 2.5 μg/mL asbestos (chrysotile, amosite, and crocidolite). Global DNA and RNA (hydroxy)methylation to cytosines was measured by a validated liquid chromatography tandem-mass spectrometry method. Global RNA methylation to adenines was measured by a colorimetric ELISA-like assay. Gene-specific DNA methylation status at certain cytosine-phosphate-guanine (CpG) sites of cyclin-dependent kinase inhibitor 1A ( CDKN1A), serine/threonine kinase ( ATM), and TNF receptor-associated factor 2 ( TRAF2) were analyzed by using bisulfite pyrosequencing technology. Only MWCNT-exposed cells showed significant global DNA hypomethylation of cytosine and global RNA hypomethylation of adenosine. SWCNT, MWCNT, and amosite exposure decreased DNA methylation of CDKN1A. ATM methylation was affected by chrysotile, SWCNT, and MWCNT. However, SWCNT exposure led to DNA hypermethylation of TRAF2. These findings contribute to further understanding of the effect of CNTs on different carcinogenic pathways.

Exploring determinants of patient behaviour based on the Theoretical Domains Framework which affect adherence to prescribed medication: A systematic review

An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of saving time and effort but exhibiting high performance, was developed using orientation-directed half-part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to detect the presence of a target substance. However, it takes time to quantify the target with specificity and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a quantum dot through a unique thiol site to properly display the recognition domain for the core process of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6 detection, as the quantification of IL-6 is significant owing to its close relationships with various biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05 ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay.

Development of ELISA-Like Fluorescence Assay for Melamine Detection Based on Magnetic Dummy Molecularly Imprinted Polymers

We present a directly competitive fluorescence assay for highly sensitive detection of melamine in milk using magnetic dummy molecularly imprinted polymers (MDMIPs). The detection principle is based on competitive binding between the fluorescent label and melamine on the MDMIPs. The fluorescent label was obtained by combining fluorescein isothiocyanate (FITC) with melamine in ethanol and water. MDMIPs were prepared on the surface of Fe3O4@SiO2 nanoparticles using 2,4-diamino-6-methyl-1,3,5-triazine as dummy template. The MDMIPs were characterized and their adsorption capacity was evaluated based on their static adsorption and Scatchard analysis. Results suggest that MDMIPs were successfully coated on the Fe3O4@SiO2 surface and had a core–shell structure. Adsorption experiments suggested that the MDMIPs had higher specific recognition capacities for melamine and FITC–melamine (FITC-Mel) than did magnetic dummy molecularly non-imprinted polymers. Competitive binding between FITC-Mel and melamine was performed under the optimum conditions to determine melamine quantitatively. The linear range of this fluorescence assay was 0.1–20 mg/L for melamine detection. The detection limit was 0.05 mg/L in negative milk samples. The assay was also successfully employed to detect melamine in spiked milk samples, with satisfactory recoveries, i.e., between 70.2% and 92.7%.

Bispecific Antibody-Functionalized Upconversion Nanoprobe.

Upconversion nanoparticles (UCNPs) are new optical probes for biological applications. For specific biomolecular recognition to be realized for diagnosis and imaging, the key lies in developing a stable and easy-to-use bioconjugation method for antibody modification. Current methods are not yet satisfactory regarding conjugation time, stability, and binding efficiency. Here, we report a facile and high-yield approach based on a bispecific antibody (BsAb) free of chemical reaction steps. One end of the BsAb is designed to recognize methoxy polyethylene glycol-coated UCNPs, and the other end of the BsAb is designed to recognize the cancer antigen biomarker. Through simple vortexing, BsAb-UCNP nanoprobes form within 30 min and show higher (up to 54%) association to the target than that of the traditional UCNP nanoprobes in the ELISA-like assay. We further demonstrate its successful binding to the cancer cells with high efficiency and specificity for background-free fluorescence imaging under near-infrared excitation. This method suggests a general approach broadly suitable for functionalizing a range of nanoparticles to specifically target biomolecules.