A microarray format for multi-parameter blood group serology

In this thesis multiple factors affecting probe-ligand interactions in a microarray format were investigated using blood group antigen and antibody sets for the development of a microarray based serology platform. Initial experiments focused on multiple parameters affecting immobilisation and functionality of monoclonal and polyclonal antibodies on various microarray surfaces. A range of critical parameters affecting protein-ligand interactions, such as efficient blocking of non-specific binding, detergent type, RBC concentration, reaction volume and mixing, were optimised in a series of experiments with labelled anti-species antibodies. Selected microarray surface and optimised spotting and reaction conditions were then used for studies of antibody-RBC antigen interactions in situ. A 'dual' solid-phase approach was investigated for blood group serology reactions where probe antibodies were immobilised on the microarray and target antigens were carried on the RBC surface, which can be considered as the second solid-phase. In summary this investigation has 1) established a microarray format and the reaction conditions for antibody-antigen interaction studies in blood group serology; 2) for the first time successfully exploited microarray format for comprehensive blood typing; 3) examined the novel technique of membrane fragment microarray immobilisation for a reverse, antibody screen reaction; 4) verified the findings and quantitatively characterised the interactions on Biacore, a surface plasmon resonance real-time detection system. This provides a strong basis for the development of microarray based multi-parameter blood group serology diagnostic platforms.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Antibodies to Enteroviruses in Cerebrospinal Fluid of Patients with Acute Flaccid Myelitis.

Acute flaccid myelitis (AFM) has caused motor paralysis in >560 children in the United States since 2014. The temporal association of enterovirus (EV) outbreaks with increases in AFM cases and reports of fever, respiratory, or gastrointestinal illness prior to AFM in >90% of cases suggest a role for infectious agents. Cerebrospinal fluid (CSF) from 14 AFM and 5 non-AFM patients with central nervous system (CNS) diseases in 2018 were investigated by viral-capture high-throughput sequencing (VirCapSeq-VERT system). These CSF and serum samples, as well as multiple controls, were tested for antibodies to human EVs using peptide microarrays. EV RNA was confirmed in CSF from only 1 adult AFM case and 1 non-AFM case. In contrast, antibodies to EV peptides were present in CSF of 11 of 14 AFM patients (79%), significantly higher than controls, including non-AFM patients (1/5 [20%]), children with Kawasaki disease (0/10), and adults with non-AFM CNS diseases (2/11 [18%]) (P = 0.023, 0.0001, and 0.0028, respectively). Six of 14 CSF samples (43%) and 8 of 11 sera (73%) from AFM patients were immunoreactive to an EV-D68-specific peptide, whereas the three control groups were not immunoreactive in either CSF (0/5, 0/10, and 0/11; P = 0.008, 0.0003, and 0.035, respectively) or sera (0/2, 0/8, and 0/5; P = 0.139, 0.002, and 0.009, respectively).IMPORTANCE The presence in cerebrospinal fluid of antibodies to EV peptides at higher levels than non-AFM controls supports the plausibility of a link between EV infection and AFM that warrants further investigation and has the potential to lead to strategies for diagnosis and prevention of disease

Upconverting diagnostics: Multiplex assays utilizing upconversion luminescence technology

Conventional diagnostics tests and technologies typically allow only a single analysis and result per test. The aim of this study was to propose robust and multiplex array-inwell test platforms based on oligonucleotide and protein arrays combining the advantages of simple instrumentation and upconverting phosphor (UCP) reporter technology. The UCPs are luminescent lanthanide-doped crystals that have a unique capability to convert infrared radiation into visible light. No autofluorescence is produced from the sample under infrared excitation enabling the development of highly sensitive assays. In this study, an oligonucleotide array-in-well hybridization assay was developed for the detection and genotyping of human adenoviruses. The study provided a verification of the advantages and potential of the UCP-based reporter technology in multiplex assays as well as anti-Stokes photoluminescence detection with a new anti- Stokes photoluminescence imager. The developed assay was technically improved and used to detect and genotype adenovirus types from clinical specimens. Based on the results of the epidemiological study, an outbreak of adenovirus type B03 was observed in the autumn of 2010. A quantitative array-in-well immunoassay was developed for three target analytes (prostate specific antigen, thyroid stimulating hormone, and luteinizing hormone). In this study, quantitative results were obtained for each analyte and the analytical sensitivities in buffer were in clinically relevant range. Another protein-based array-inwell assay was developed for multiplex serodiagnostics. The developed assay was able to detect parvovirus B19 IgG and adenovirus IgG antibodies simultaneously from serum samples according to reference assays. The study demonstrated that the UCPtechnology is a robust detection method for diverse multiplex imaging-based array-inwell assays.Perinteiset diagnostiset testit mahdollistavat vain yhden analyysin ja tuloksen määritystä kohden. Väitöskirjatyöni tavoitteena oli kehittää uudenlaisia järjestelmä kuopassa tyyppisiä mallintavia monianalyyttimäärityksiä, joissa yhdestä näytteestä yhdessä reaktiokaivossa voidaan mitata samanaikaisesti useita analyyttejä ja niiden pitoisuuksia. Kehitetyt monianalyyttimääritykset perustuvat sekä nukleiinihappojen että proteiinien mittaukseen hyödyntäen leimoina valoa tuottavia epäorgaanisia lantanidi-ioneja sisältäviä loisteainepartikkeleita (UCP). Näillä partikkeleilla on ainutlaatuinen kyky virittyä matalaenergisen infrapunavalon vaikutuksesta ja emittoida korkeaenergisen näkyvän valon aallonpituuksilla, mikä mahdollistaa autofluoresenssista vapaan mittauksen ja näin ollen herkkien määrityksien kehityksen. Väitöskirjatyössäni kehitin adenoviruksille DNA-tyypitysmäärityksen, joka perustui yksinauhaisen PCR-tuotteen ja adenovirus-tyyppispesifisten koettimien väliseen hybridisaatioreaktioon mikrotiitterilevyn kaivon pohjalla. Tulokset osoittivat, että UCP-leimateknologia ja uusi anti-Stokes luminesenssin kuvantamiseen kehitetty laite soveltuvat monianalyttimääritysten kvantitatiiviseen mittaukseen. Teknisesti parannettua adenovirusten DNA-tyypitysmenetelmää hyödynnettiin kliinisesti merkittävien adenovirustyyppien havaitsemisessa ja genotyypityksessä. Adenovirusinfektioiden epidemiologinen seuranta osoitti, että adenovirus tyyppi B03 aiheutti epidemian syksyllä 2010. Väitöskirjatyössäni kehitin myös kvantitatiivisen mallintavan monianalyytti-immunomäärityksen kolmelle antigeenille. Määrityksessä kaikille analyyteille saatiin kvantitatiivinen tulos, ja analyyttiset herkkyydet olivat kliinisesti merkittävällä alueella. Lisäksi väitöskirjatyössäni kehitin serologisen monianalyyttimäärityksen, jonka avulla mitattiin seerumista IgG-luokan adenovirusja parvovirus B19-vasta-aineita samanaikaisesti. Serologinen monianalyyttimääritys validoitiin näytepaneelilla ja tulokset korreloivat hyvin adenovirus IgG ja parvovirus B19 IgG referenssi-entsyymi-immunomäärityksien kanssa. Väitöskirjatyössäni osoitin, että UCP-teknologia soveltuu sekä oligonukleotidi- että proteiini-pohjaisten monianalyyttimääritysten kvantitatiiviseen kuvantamiseen.Siirretty Doriast

Review Updated Values for Molecular Diagnosis for Highly Pathogenic Avian Influenza Virus

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Accuracy of Blood Group Typing in the Management and Prevention of Alloimmunization

Blood transfusion is an effective therapeutic approach for several hematological conditions including sickle cell disease (SCD), thalassaemia, myelodysplastic syndrome (MDS), and autoimmune hemolytic anemia. It is also often indicated for transplantation and for patients receiving medical treatments for cancer. However, transfusion treatment can lead to the red blood cell (RBC) alloimmunization when an incompatible antigen is inadvertently present in the transfused blood. Alloantibodies can cause RBC destruction and many other complications defeating the purpose of the treatment. The risk of development of multiple alloantibodies increases with the frequency of transfusions in transfusion-dependent patients and can be mitigated by transfusing blood type negative for multiple antigens to prevent hemolysis. This chapter discusses the transfusion’s risk of RBC alloimmunization as an adverse event; consequences of alloimmunization in patients’ care; approaches to prevent and/or mitigate alloimmunization and enhance transfusion efficacy; application of RBC genotyping to supplement serology for preventing alloimmunization. The currently available techniques for RBC genotyping and the importance of reference reagents for determining the genotyping accuracy will also be discussed

Identifying Influenza Viruses with Resequencing Microarrays

Resequencing microarrays rapidly identify influenza viruses

레이저 활성 세포 분리 기기를 이용한 조직 내 세포 분리 및 전장 유전체 및 전사체 분석 기술 개발

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2020. 8. 권성훈.In this dissertation, Spatially-resolved Laser Activated Cell Sorting (SLACS) technique is introduced, and its applications in genomics and transcriptomics are demonstrated. All biological mass is comprised of biological cells, each of which contain its own multi-billion bytes worth of data from genetic molecules, such as DNA or RNA. After the Human Genome Project sequenced one persons genome in ten years, the massively parallel sequencing technologies that are referred to the next generation sequencing (NGS) sprouted innovations in biology, providing further insights into biology and generating revolutions in diagnostics and therapeutics. However, these technologies were only applicable to pools of heterogeneous genetic molecules, hindering thorough explorations of genetic landscapes in the different cells within a biospecimen. Therefore, efforts to separate each and every cell from the pool of cells have generated numerous single cell isolation methodologies, which can be categorized into three: those that separate cell using microfluidics, microarrays, and optics. Advancement in micro-technologies particularly provided advantages in manipulating single cells because biological cell sizes that usually range from microns to tens of microns. State-of-art cell separation technologies that utilize microfluidic properties were rapidly commercialized, enabling high throughput single cell analysis that can process hundreds to thousands of single cells at a time. These utilize cell dissociation and compartmentalization in a microfluidic chambers or a pico-liter droplets, in which biomolecular techniques can amplify the desired genetic molecules. The amplified products such as the genomes or the transcriptomes of the single cells are sequenced through NGS, providing insights into how the dissociated cells were functioning in the biospecimen. However, the dissociation process of the cells that are originally adhered to each other can be harsh and requires the surface proteins that interact with another to be degraded. This process has raised many doubts on whether the cell state is the same before it is dissociated within a solvent. Therefore, microarrays of chemically synthesized oligonucleotides that can capture the poly adenosine tail, or poly (A) tail, were developed to capture the messenger RNAs (mRNAs) directly from the biological specimens. These technologies, however, require large resolution of the oligonucleotide spots because of the technical limitations in chemical DNA synthesis technologies and cross-contaminations between the spots. Optical separation of the cells from biospecimen has been extensively investigated with conventional laser capture microdissection (LCM) devices that utilize laser to transfer target area of interest to the desired receiver. However, these utilize either ultraviolet (UV) lasers to catapult the desired areas that can be highly damaging to the biomolecules within, or thermoplastics that can be melt down using near-infrared (IR) lasers and transfer the desired region of interest for further biological assays. However the thermoplastic approach often cause cross-contamination and has low throughput because the specimen has to be isolated in a contact manner. In this dissertation, the development of an optical cell sorter, or spatially-resolved laser activated cell sorter (SLACS) that uses pulsed near-IR laser that can optomechanically isolate the cells with low damage and high throughput is described. The engineering process of this novel device and two softwares and their applications in NGS technologies are described. Furthermore, the applications of SLACS for genomics and transcriptomics are demonstrated. Proof-of-concept studies for future applications of SLACS are also described.본 학위 논문에서는 이 논문에서는 SLACS (Spatially-resolved Laser Activated Cell Sorting) 기술이 도입되었으며 유전체학 및 전사체학에 대한 응용이 시연되었다. 모든 생물학적 덩어리는 생물학적 세포로 구성되며, 각각의 세포는 DNA 또는 RNA와 같은 유전자 분자로부터 얻은 수십억 바이트의 데이터를 포함한다. 휴먼 게놈 프로젝트가 10 년 안에 한 사람의 게놈을 시퀀싱 한 후, 차세대 시퀀싱 (NGS)과 관련되는 대규모 병렬 시퀀싱 기술은 생물학의 혁신을 불러 일으켜 생물학에 대한 통찰력을 제공하고 진단 및 치료에서 혁명을 일으켰다. 그러나 이 기술들은 이종 유전자 분자의 풀에만 적용 할 수 있으며, 생물 표본 내의 다른 세포에서 유전자 지형의 철저한 탐색을 방해하였다. 따라서, 세포 풀에서 각각의 모든 세포를 따로 분리하려는 노력은 수많은 단일 세포 분리 방법론을 생성하였으며, 이는 미세유체학, 마이크로 어레이 및 광학을 사용하여 세포를 분리하는 것의 세 가지로 분류 될 수 있다. 일반적으로 수 마이크로미터에서 수십 마이크로미터에 이르는 생물학적 세포 크기 때문에 단일 기술의 진보는 단일 세포 조작에 이점을 제공 하였다. 미세 유체 특성을 이용하는 최첨단 세포 분리 기술이 빠르게 상용화되어 한 번에 수백에서 수천 개의 단일 세포를 처리 할 수 있는 고 처리량 단일 세포 분석이 가능해졌습니다. 이들은 미세 분자 챔버 또는 피코 리터 액적에서 세포 해리 및 구획화를 이용하며, 여기서 생체 분자 기술은 원하는 유전자 분자를 증폭시킬 수 있다. 단일 세포의 게놈 또는 전 사체와 같은 증폭 된 생성물은 NGS를 통해 시퀀싱되어, 해리 된 세포가 생체 시편에서 어떻게 기능하는지에 대한 통찰력을 제공한다. 그러나, 원래 서로 부착 된 세포의 해리 과정은 가혹할 수 있으며, 서로 상호 작용하는 표면 단백질이 분해 될 것을 요구한다. 이 공정은 전지 상태가 용매 내에서 해리되기 전에 동일한 지에 대해 많은 의문을 제기했다. 따라서, 폴리아데노신 꼬리 또는 폴리 (A) 꼬리를 포획 할 수 있는 화학적으로 합성 된 올리고 뉴클레오티드의 마이크로 어레이는 생물학적 표본으로부터 메신저 RNA (mRNA)를 직접 포획하기 위해 개발되었다. 그러나, 이들 기술은 화학적 DNA 합성 기술의 기술적 한계 및 스폿 간의 교차 오염으로 인해 올리고 뉴클레오티드 스폿의 큰 해상도를 요구한다. 생체 시료로부터 세포의 광학적 분리는 관심 대상 영역을 원하는 수신기로 전달하기 위해 레이저를 이용하는 종래의 레이저 캡처 미세 해부 (LCM) 장치로 광범위하게 조사되었다. 그러나, 이들은 자외선 (UV) 레이저를 사용하여 생체 내 분자에 크게 손상을 줄 수있는 원하는 영역을 만들거나 근적외선 (IR) 레이저를 사용하여 녹일 수 있고 추가 생물학적 물질을 위해 원하는 관심 영역을 전달할 수있는 열가소성 수지를 사용한다. 분석. 그러나 열가소성 방식은 종종 교차 오염을 유발하고 시편을 접촉 방식으로 분리해야하기 때문에 처리량이 낮다. 이 논문에서는 광학 셀 분류기 또는 낮은 손상과 높은 처리량으로 셀을 광학적으로 분리 할 수 있는 펄스 형 근적외선 레이저를 사용하는 SLACS (공간적으로 해결 된 레이저 활성화 셀 분류기)의 개발에 대해 설명하였다. 이 새로운 장치의 엔지니어링 프로세스와 NGS 기술의 두 소프트웨어 및 응용 프로그램에 대해 설명하였다. 또한, 게놈 및 전 사체에 대한 SLACS의 적용이 입증되었다. SLACS의 향후 응용에 대한 개념 증명 연구도 설명하였다.CHAPTER 1. INTRODUCTION １ 1.1. Spatially resolved omics for atlasing human cells in the biological circuitry ２ 1.1.1. The emergence of single cell sequencing technologies ３ 1.1.2. Spatially resolved omics technologies and needs for development ７ 1.2. Main Concept: Development of spatially-resolved laser activated cell sorter (SLACS) and compatible omics technologies １４ 1.3. Outline of the dissertation １５ CHAPTER 2. BACKGROUND １６ 2.1. Previous spatial omics technologies １７ 2.1.1. In situ spatial omics technologies １７ 2.1.2. Isolate-and-transfer technologies for spatial omics ２０ 2.2. Commercialized spatial omics technologies ２３ 2.3. Previous research in the group ２５ CHAPTER 3. PLATFORM DEVELOPMENT ２９ 3.1. Development of SLACS and remote selection system ３０ 3.2. Whole genome sequencing strategies for SLACS ３３ 3.3. Whole transcriptome sequencing strategies for SLACS ４２ CHAPTER 4. PLATFORM APPLICATION ４８ 4.1. Applications of SLACS to spatial genomics ４９ 4.2. Applications of SLACS to spatial transcriptomics ６２ 4.3. Applications of OPENchip and future perspectives with SLACS ６５ CHAPTER 5. CONCLUSION AND DISCUSSION ７９ 5.1. Summary of dissertation ８０ 5.2. Comparison with previous technology ８３ 5.3. Limit of the platform ８４ 5.4. Future work ８６ BIBLIOGRAPHY ８８ 국문 초록 ９５Docto

Recent trends in molecular diagnostics of yeast infections : from PCR to NGS

The incidence of opportunistic yeast infections in humans has been increasing over recent years. These infections are difficult to treat and diagnose, in part due to the large number and broad diversity of species that can underlie the infection. In addition, resistance to one or several antifungal drugs in infecting strains is increasingly being reported, severely limiting therapeutic options and showcasing the need for rapid detection of the infecting agent and its drug susceptibility profile. Current methods for species and resistance identification lack satisfactory sensitivity and specificity, and often require prior culturing of the infecting agent, which delays diagnosis. Recently developed high-throughput technologies such as next generation sequencing or proteomics are opening completely new avenues for more sensitive, accurate and fast diagnosis of yeast pathogens. These approaches are the focus of intensive research, but translation into the clinics requires overcoming important challenges. In this review, we provide an overview of existing and recently emerged approaches that can be used in the identification of yeast pathogens and their drug resistance profiles. Throughout the text we highlight the advantages and disadvantages of each methodology and discuss the most promising developments in their path from bench to bedside

Chagas Disease Diagnostic Applications: Present Knowledge and Future Steps

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a lifelong and debilitating illness of major significance throughout Latin America and an emergent threat to global public health. Being a neglected disease, the vast majority of Chagasic patients have limited access to proper diagnosis and treatment, and there is only a marginal investment into R&D for drug and vaccine development. In this context, identification of novel biomarkers able to transcend the current limits of diagnostic methods surfaces as a main priority in Chagas disease applied research. The expectation is that these novel biomarkers will provide reliable, reproducible and accurate results irrespective of the genetic background, infecting parasite strain, stage of disease, and clinical-associated features of Chagasic populations. In addition, they should be able to address other still unmet diagnostic needs, including early detection of congenital T. cruzi transmission, rapid assessment of treatment efficiency or failure, indication/prediction of disease progression and direct parasite typification in clinical samples. The lack of access of poor and neglected populations to essential diagnostics also stresses the necessity of developing new methods operational in point-of-care settings. In summary, emergent diagnostic tests integrating these novel and tailored tools should provide a significant impact on the effectiveness of current intervention schemes and on the clinical management of Chagasic patients. In this chapter, we discuss the present knowledge and possible future steps in Chagas disease diagnostic applications, as well as the opportunity provided by recent advances in high-throughput methods for biomarker discovery.Fil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Fernandez Aguero, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin