Development of impedimetric immunosensors for the detection of a range of antigens of biological or biomedical significance

This PhD project was funded by the European Union Framework 6 ELISHA programme as part of the ELISHA programme. The aim of the research was focused towards the development of point-of-care, simple, cost-efficient and reliable impedimetric immunosensors for the rapid detection of important antigens such as ciprofloxacin and digoxin. Through the cooperation of the 9 involved partners a number of protocols have been developed for sensor fabrication and sample testing that allow both rapid and reliable detection of a range of antigens. This work describes in depth, the use of polymers and cyclic voltammetry for electrode surface modification, the use of the avidin-biotin system for antibody immobilisation and finally the use of Electrochemical Impedance Spectroscopy for antigen detection. We report here the successful development of electrochemical impedimetric carbon based immunosensors for the detection of free-form and chelated ciprofloxacin (both in laboratory buffer and milk), digoxin and green fluorescent protein. It was observed during this work that unavailability of sufficient quantities of the monoclonal antibodies could lead to early sensor saturation and hence a less extended antigen detection range, while very low quantities of immobilised antibodies could also give rise to erroneous results. The immunosensors towards ciprofloxacin detected the respective antigen when this was present between 1 ng ml-1 and 10 μg ml-1 in PBS buffer and 0.1 ng ml-1 to 10 μg ml-1 when the antigen was added to milk samples. The developed digoxin immunosensors could detect digoxin between the concentrations of 0.1 ng ml-1 and 10 μg ml-1. Lower detection limits where observed for the sensors targeting GFP which could detect their respective antigen at concentrations as low as 100 pg ml-1. The tested concentration range of the latter sensors was extended up to 100 ng ml-1

Development of impedimetric immunosensors for the detection of a range of antigens of biological or biomedical significance

This PhD project was funded by the European Union Framework 6 ELISHA programme as part of the ELISHA programme. The aim of the research was focused towards the development of point-of-care, simple, cost-efficient and reliable impedimetric immunosensors for the rapid detection of important antigens such as ciprofloxacin and digoxin. Through the cooperation of the 9 involved partners a number of protocols have been developed for sensor fabrication and sample testing that allow both rapid and reliable detection of a range of antigens. This work describes in depth, the use of polymers and cyclic voltammetry for electrode surface modification, the use of the avidin-biotin system for antibody immobilisation and finally the use of Electrochemical Impedance Spectroscopy for antigen detection. We report here the successful development of electrochemical impedimetric carbon based immunosensors for the detection of free-form and chelated ciprofloxacin (both in laboratory buffer and milk), digoxin and green fluorescent protein. It was observed during this work that unavailability of sufficient quantities of the monoclonal antibodies could lead to early sensor saturation and hence a less extended antigen detection range, while very low quantities of immobilised antibodies could also give rise to erroneous results. The immunosensors towards ciprofloxacin detected the respective antigen when this was present between 1 ng ml-1 and 10 μg ml-1 in PBS buffer and 0.1 ng ml-1 to 10 μg ml-1 when the antigen was added to milk samples. The developed digoxin immunosensors could detect digoxin between the concentrations of 0.1 ng ml-1 and 10 μg ml-1. Lower detection limits where observed for the sensors targeting GFP which could detect their respective antigen at concentrations as low as 100 pg ml-1. The tested concentration range of the latter sensors was extended up to 100 ng ml-1.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Spread of Vector-borne Diseases and Neglect of Leishmaniasis, Europe

Exotic vector-borne diseases are gaining attention at the expense of leishmaniasis

Sonochemically Fabricated Microelectrode Arrays for Use as Sensing Platforms

The development, manufacture, modification and subsequent utilisation of sonochemically-formed microelectrode arrays is described for a range of applications. Initial fabrication of the sensing platform utilises ultrasonic ablation of electrochemically insulating polymers deposited upon conductive carbon substrates, forming an array of up to 70,000 microelectrode pores cm−2. Electrochemical and optical analyses using these arrays, their enhanced signal response and stir-independence area are all discussed. The growth of conducting polymeric “mushroom” protrusion arrays with entrapped biological entities, thereby forming biosensors is detailed. The simplicity and inexpensiveness of this approach, lending itself ideally to mass fabrication coupled with unrivalled sensitivity and stir independence makes commercial viability of this process a reality. Application of microelectrode arrays as functional components within sensors include devices for detection of chlorine, glucose, ethanol and pesticides. Immunosensors based on microelectrode arrays are described within this monograph for antigens associated with prostate cancer and transient ischemic attacks (strokes)

CSF-1 maintains pathogenic but not homeostatic myeloid cells in the central nervous system during autoimmune neuroinflammation

SignificanceMultiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by accumulation of myeloid cells in the central nervous system (CNS). Both harmful and beneficial myeloid cells are present in EAE/MS, and a goal of MS therapy is to preferentially remove harmful myeloid cells. The receptor for CSF-1 (CSF-1R) is found on myeloid cells and is important for their survival. CSF-1R can bind two ligands, CSF-1 and IL-34, but it is not known whether their functions in EAE/MS differ. We found that blocking CSF-1 depleted only harmful myeloid cells in the CNS and suppressed EAE, whereas blocking IL-34 had no effect. Thus, we propose that blocking CSF-1 could be a therapy for MS

Molecular Characterization of Leishmania Species Isolated from Cutaneous Leishmaniasis in Yemen

Background: Cutaneous leishmaniasis (CL) is a neglected tropical disease endemic in the tropics and subtropics with a global yearly incidence of 1.5 million. Although CL is the most common form of leishmaniasis, which is responsible for 60% of DALYs lost due to tropical-cluster diseases prevalent in Yemen, available information is very limited. Methodology/Principal Findings: This study was conducted to determine the molecular characterization of Leishmania species isolated from human cutaneous lesions in Yemen. Dermal scrapes were collected and examined for Leishmania amastigotes using the Giemsa staining technique. Amplification of the ribosomal internal transcribed spacer 1(ITS-1) gene was carried out using nested PCR and subsequent sequencing. The sequences from Leishmania isolates were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods. The trees identified Leishmania tropica from 16 isolates which were represented by two sequence types. Conclusions/Significance: The predominance of the anthroponotic species (i.e. L. tropica) indicates the probability of anthroponotic transmission of cutaneous leishmaniasis in Yemen. These findings will help public health authorities to build an effective control strategy taking into consideration person–to-person transmission as the main dynamic of transmissio

IL-11 Induces NLRP3 Inflammasome Activation in Monocytes and Inflammatory Cell Migration to the Central Nervous System

The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the central nervous system (CNS). We report that IL-11 is produced at highest frequency by myeloid cells among the peripheral blood mononuclear cell (PBMC) subsets. Patients with relapsing-remitting multiple sclerosis (RRMS) have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes, and IL-11R+ neutrophils in comparison to matched healthy controls. IL-11+ and granulocyte-macrophage colony-stimulating factor (GM-CSF)+ monocytes, CD4+ lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single-cell RNA sequencing, revealed the highest number of differentially expressed genes in classical monocytes, including up-regulated NFKB1, NLRP3, and IL1B. All CD4+ cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation. In IL-11R+-sorted cells from the CSF, classical and intermediate monocytes significantly up-regulated the expression of multiple NLRP3 inflammasome-related genes, including complement, IL18, and migratory genes (VEGFA/B) in comparison to blood-derived cells. Therapeutic targeting of this pathway with αIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates, and demyelination. αIL-11 mAb treatment decreased the numbers of NFκBp65+, NLRP3+, and IL-1β+ monocytes in the CNS of mice with EAE. The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS

Concept and design of a genome-wide association genotyping array tailored for transplantation-specific studies

Background: In addition to HLA genetic incompatibility, non-HLA difference between donor and recipients of transplantation leading to allograft rejection are now becoming evident. We aimed to create a unique genome-wide platform to facilitate genomic research studies in transplant-related studies. We designed a genome-wide genotyping tool based on the most recent human genomic reference datasets, and included customization for known and potentially relevant metabolic and pharmacological loci relevant to transplantation. Methods: We describe here the design and implementation of a customized genome-wide genotyping array, the ‘TxArray’, comprising approximately 782,000 markers with tailored content for deeper capture of variants across HLA, KIR, pharmacogenomic, and metabolic loci important in transplantation. To test concordance and genotyping quality, we genotyped 85 HapMap samples on the array, including eight trios. Results: We show low Mendelian error rates and high concordance rates for HapMap samples (average parent-parent-child heritability of 0.997, and concordance of 0.996). We performed genotype imputation across autosomal regions, masking directly genotyped SNPs to assess imputation accuracy and report an accuracy of >0.962 for directly genotyped SNPs. We demonstrate much higher capture of the natural killer cell immunoglobulin-like receptor (KIR) region versus comparable platforms. Overall, we show that the genotyping quality and coverage of the TxArray is very high when compared to reference samples and to other genome-wide genotyping platforms. Conclusions: We have designed a comprehensive genome-wide genotyping tool which enables accurate association testing and imputation of ungenotyped SNPs, facilitating powerful and cost-effective large-scale genotyping of transplant-related studies. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0211-x) contains supplementary material, which is available to authorized users