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Narmack, Samuel. "Functionalization and Evaluation of Nanoparticle Probes for the Development of a 14-Plex Diagnostic assay." Thesis, KTH, Kemi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-299949.
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Detta projekt var ett samarbete mellan Aplex Bio AB och Scilifelab. Projektets mål var att utveckla en molekylär diagnostisk panel med förmågan att detektera och diskriminera mellan 14 olika typer av patogener. Projektet innehåller 4 kapitel med fokus på olika mål. I första kapitlet utvecklades en metod för att karakterisera emissioner av fluorescerande nanopartikel kluster. Den första utvärderade metoden utnyttjade klick-kemi för att binda nanopartiklarna till makrostrukturer uppbyggda av amplifierat DNA. Den andra utvärderade metoden skapade aggregerade komplex av nanopartiklar med amplifierat DNA för att utvärdera partiklarnas emissioner. I kapitel 2 av projektet användes azid-funktionaliserade nanopartiklar levererade av Aplex Bio AB för att tvärbinda DBCO modifierade oligonukleotider. Sedan utvecklades en hybridiserings baserad metod för att kvantifiera relativa mängden oligonukleotider på partiklarna. Denna metod användes för att reproducerbart funktionalisera partiklar och utveckla nanopartikel-sonder som kan binda till DNA genom hybridisering. I kapitel 2 utvärderades även hur effektivt och specifikt de utvecklade nanopartikel-sonderna hybridiserar till DNA. I kapitel 3 utvärderades amplifiering av syntetiska ssDNA sekvenser valda från genetiska markörer av 14 patogener, DNA amplifierades med metoden RCA. Målet var att utvärdera specificiteten av amplifieringen. Specifik amplifiering av varje DNA sekvens i panelen var en förutsättning för att möjliggöra detektion och diskriminering av alla patogener i panelen. I kapitel 4 var målet att utveckla en kostnadseffektiv metod för att funktionalisera nanopartiklar med oligonukleotid sekvenser. För att göra detta användes DBCO-NHS-ester reagens och amin-modifierade oligonukleotider. Förverkligande av detta projekt skulle skapa en diagnostisk panel med potentialen att påverka det diagnostiska fältet på en global skala. När detta projekt är fullt utvecklat kan panelen modifieras för detektion av önskade DNA/RNA sekvenser vilket möjliggör ett mångfalt av applikationer, detta skulle göra panelen konkurrerande med dagens diagnostiska metoder då den kan användas i existerande mikroskopiuppsättningar.<br>This work was a collaboration between Aplex Bio AB and Scilifelab with the aim of developing a molecular assay capable of detecting and discriminating between 14 different pathogenic targets. There are 4 chapters with focus on different goals. In chapter one a method of evaluating emissions of fluorescent nanoparticle clusters was developed. The first approach of evaluating nanoparticle emissions was to utilize click chemistry to bind nanoparticles to macroscale structures of amplified DNA targets. The second evaluated approach was the formation of aggregated complexes of nanoparticles and amplified DNA targets. The second chapter of the thesis used azide functionalized nanoparticles supplied by Aplex Bio AB to utilize azide groups as crosslinkers and use them to functionalize the nanoparticles with DBCO oligos. A hybridization-based method was then developed to quantify relative oligo densities on the nanoparticles, enabling reproducible oligo functionalization of nanoparticles, producing nanoparticle probes that can bind to DNA. The final task of chapter 2 was evaluating the binding efficiency and specificity of the developed nanoparticle probes. The third chapter of the thesis evaluated amplification of synthetic ssDNA sequences corresponding to genetic markers of 14 pathogenic targets using RCA. The goal was to confirm specificity of chosen padlock probes and corresponding synthetic targets for each pathogen. Specific amplification of each target was a prerequisite to enable detecting and discriminating between the 14 pathogenic targets. In chapter 4 the goal was to develop a cost-effective method of oligo functionalization for nanoparticles. This chapter evaluated two main approaches of using DBCO-NHS-ester reagents to perform DBCO modification of amine-oligos. The realization of this work would develop an assay that has the potential to impact the field of diagnostics on a global scale. When fully developed, the molecular assay can be modified to detect any RNA/DNA targets which enables numerous applications, making the assay a competitive diagnostic tool which can be implemented in existing microscopy systems.
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Wan, Zhuohua. "Newly recognized rat parvoviruses : characterization and diagnostic assay development /." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3013040.
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Frazenburg, Lolita. "The development of a diagnostic assay for nepoviruses in grapevine." Thesis, Stellenbosch : Stellenbosch University, 2015. http://hdl.handle.net/10019.1/96964.
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Thesis (MSc)--Stellenbosch University, 2015.<br>ENGLISH ABSTRACT: The nepoviruses are a group of nematode-transmitted plant viruses that are
distributed worldwide and infect a wide range of plant species, including grapevine.
Most of the nepoviruses are foreign to South Africa and to date, only Grapevine
fanleaf virus (GFLV) is present. The Department of Agriculture, Forestry and
Fisheries (DAFF), as the official National Plant Protection Organisation (NPPO) of
South Africa, is committed to prevent the importation and spread of plant pathogens
by administering the Agricultural Pests Act, 1983 (Act No. 36 of 1983). Effective
measures are implemented by which the introduction of agricultural pests may be
prohibited to safeguard the agricultural environment. One of the core functions of
DAFF is to render a routine plant health diagnostic service for imported plants and
plant products to prevent exotic pathogens from entering the country. The objective
of this study was to develop a diagnostic assay for the detection of nepoviruses in
grapevine. The project aimed to produce antibodies by recombinant DNA technology
against bacterially expressed viral coat protein of a specific nepovirus [Tomato
ringspot virus (ToRSV)] and subsequently develop a DAS-ELISA (Double Antibody
Sandwich Enzyme-linked Immunosorbent) assay for the detection of the virus. The
coat protein (CP) was successfully isolated from imported ToRSV-infected grapevine
material. Two expression systems were utilised for expression of the ToRSV-CP, the
GST gene fusion system and an Agrobacterium-mediated expression system. The
GST gene fusion system was unsuccessful as insufficient soluble protein expression
prevented the production of antibodies and thus the development of the DAS-ELISA
assay. Tissue print immunoassay (TPIA) initially showed positive results for transient
expression of the fusion protein in tobacco plants, but further confirmation proved to
be inconclusive. The project also aimed to develop a real-time PCR assay for the
specific detection and relative quantification of GFLV, based on a conserved region
of the RNA-2 genome. A partial GFLV-RNA-2 from a South African isolate of
grapevine was sequenced and used for the design of specific primers. The
quantitative real-time PCR assay based on SYBR green technology proved to be
sensitive in detecting levels as low as 0.11ng/reaction in infected plants, making it a
highly effective diagnostic tool for the detection of GFLV.<br>AFRIKAANSE OPSOMMING: Die nepovirusse is 'n groep van nematode-oordraagbare plant virusse wat
wêreldwyd versprei word en 'n wye verskeidenheid van plantspesies infekteer,
insluitend wingerd. Die meeste van die nepovirusse is uitheems aan Suid-Afrika en
tot op datum is net Wingerd netelblaar virus (GFLV) teenwoordig. Die Departement
van Landbou, Bosbou en Visserye (DAFF), as die amptelike Nasionale Plant
Beskermings Organisasie (NPBO) van Suid-Afrika, is daartoe verbind om die invoer
en verspreiding van plantpatogene te voorkom deur administrasie van die Wet op
Landbouplae, 1983 (Wet No. 36 van 1983). Doeltreffende maatreëls word
geïmplementeer waardeur die invoer van landbouplae verbied word om sodoende
die landbou-omgewing te beskerm. Een van die kernfunksies van DAFF is om 'n
roetine plant gesondheid diagnostiese diens vir ingevoerde plante en plantprodukte
te lewer om te verhoed dat eksotiese patogene die land binnedring. Die doel van
hierdie studie was om 'n diagnostiese toets vir die opsporing van nepovirusse in
wingerd te ontwikkel. Die projek was daarop gemik om antiliggame te vervaardig
deur rekombinante DNA-tegnologie teen bakterieël-uitgedrukte virale mantelproteïen
van 'n spesifieke nepovirus [Tomato ringspot virus (ToRSV)] en vervolgens ‘n DASELISA
(Double Antibody Sandwich Enzyme-linked Immunosorbent) toets vir die
opsporing van die virus te ontwikkel. Die mantelproteïen (CP) is met sukses
geïsoleer vanaf ingevoerde ToRSV-besmette wingerdmateriaal. Twee uitdrukking
stelsels is gebruik vir uitdrukking van die ToRSV-CP, die “GST gene fusion” stelsel
en 'n Agrobacterium-bemiddelde uitdrukking stelsel. Die “GST gene fusion” stelsel
was egter onsuksesvol aangesien onvoldoende oplosbare proteïen uitdrukking die
produksie van antiliggame en dus die ontwikkeling van die DAS-ELISA toets verhoed
het. “Tissue print immunoassay” (TPIA) het aanvanklik positiewe resultate getoon vir
tydelike uitdrukking van die fusie proteïen in tabakplante, maar verdere bevestiging
was onoortuigend. Die projek was ook daarop gemik om ‘n in-tyd polimerase ketting
reaksie (PKR) toets vir die spesifieke opsporing en relatiewe kwantifisering van
GFLV, gebaseer op 'n gekonserveerde volgorde van die RNA-2 genoom, te
ontwikkel. 'n Gedeeltelike GFLV-RNA-2 nukleïensuurvolgorde van 'n Suid-Afrikaanse
wingerd isolaat is bepaal en gebruik vir die ontwerp van spesifieke inleiers. Die
kwantitatiewe in-tyd PKR toets gebaseer op SYBR groen tegnologie was sensitief
genoeg om vlakke van so laag as 0.11ng/reaksie in geïnfekteerde plante op te
spoor, wat dit 'n hoogs effektiewe diagnostiese hulpmiddel vir die opsporing van
GFLV maak.
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Fraser, Sarah Jane. "Development of a diagnostic serological assay for ovine herpesvirus-2." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/29770.
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The aim of this project was to develop a serological assay based on recombinant antigens derived from the OvHV-2 genome and validation of the ELISA test using clinical serum samples. Eight OvHV-2 genes were selected for expression in three bacterial systems. Four genes could in at least one system. Three genes were expressed in an <i>in vitro</i> transcription-translation system (Ov8, OV8.5 and orf65). Protein purification, using epitope-tag affinity purification and conventional chromatography, was unsuccessful. Due to the lack of recombinant antigen expression, crude antigen preparations from cultured AIHV-1 and OvHV-2 infected cells were analysed. Antigens suitable for the detection of MCF virus specific serum antibodies could not be extracted from an OvHV-2 infected cell line but AIHV-1 WC11 antigen could be used to differentiate MCF-positive and –negative sera. An ELISA based on this crude antigen was established, designated WC11-ELISA. Initial validation revealed over 93% concordance between WC11-ELISA and commercial ELISA, while a comparison with serological assays, a PCR test and final veterinary evaluations, showed good agreement between tests. The WC11-ELISA showed 57% agreement to PCR while commercial CI-ELISA showed 66% agreement for the same samples. This indicates the value of the ELISA as an economical, rapid and specific serological assay for the detection of antibodies against MCFV. Potentially antigenic components of the ELISA lysates were analysed by western blotting in comparison with other bovine herpesviruses, and fractionation by gel filtration showed clear antigenic bands in the eluent. Proteomic analysis of antigenic bands identified proteins encoding viral thymidine kinase (orf21), a virus capsid protein (orf25) and a viral antigen (orf54) which encodes a dUTPase.
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Gibbons, Cindy Louise. "Development of a strain specific diagnostic/detection assay for Cryptosporidium parvum." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325584.
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Affan, Noha Ahmed. "Molecular characterisation of Toxoplasma gondii and development of diagnostic assay for bradyzoites." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/13274/.
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Toxoplasma gondii (T. gondii) is a ubiquitous parasite that infects warm-blooded animals and humans. In humans, T. gondii causes encephalitis in AIDS patients, and there is no drug that can eliminate T. gondii infection. T. gondii specifically manipulates the intermediate host’s behaviour favouring its transmission to the definitive feline host. Human T. gondii seropositivity has also been associated with mental disorders. T. gondii has two aromatic amino acid hydroxylases, TgAaaH (1 and 2), that convert phenylalanine to tyrosine, and tyrosine to L- DOPA, the latter being the rate-limiting step of dopamine biosynthesis. Based on this and elevated dopamine levels in brain tissue cysts and infected dopaminergic cells, it has been hypothesised that TgAaaH has a role in altering brain neuromodulation and potentially subsequently in the behavioural changes observed. As TgAaaH genes encode a signal peptide, the location of the enzyme was examined. TgAaaH was localised to outside the parasite both membrane- bound to parasites within the parasitophorous vacuole based on immunofluorescence, fractionation, and trypsin susceptibility of released parasites. Another possible role of TgAaaH in cyst wall generation was examined by testing for dopa-oxidase activity to convert L-DOPA to dopa- quinone. Dopa-oxidase activity was not detectable in infected fibroblasts, yet it remains possible that parasite produced L-DOPA is metabolised to dopaquinone by host cell enzymes such as within feline gut endothelial cells where oocysts are formed. This, and our finding that host cell dopa-decarboxylase is required for dopamine biosynthesis, suggest that the product of parasite-produced L-DOPA may be dependent upon the type of cell infected (ie. dopamine in catecholaminergic cells and dopaquinone in endothelial cells). The limitation of studying the bradyzoite stages of infection due to proliferation of tachyzoite stages was resolved by development of a new culture system with depleted tryptophan. This method was then applied to develop a novel throughput assay to identify bradyzoite inhibitors. The validity of this assay was evaluated using tachyzoite and bradyzoite specific inhibitors. This assay will help in finding an anti-toxoplasma drug for curing of infection.
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Ebai, Tonge. "Development of Enhanced Molecular Diagnostic Tools for Protein Detection and Analysis." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-320380.
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Improved diagnosis, prognosis and disease follow-up is a fundamental procedure and a constant challenge in medicine. Among the different molecular biomarkers, proteins are the essential regulatory component in blood; hence, by developing enhanced specific and sensitive molecular tools will gives great insight into the different processes in disease treatment. In this thesis, we build on the proximity ligation assay to develop and apply new adaptable methods to facilitate protein detection. In paper I, I present a variant of the proximity ligation assay (we call PLARCA) using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer. PLARCA detected femtomolar levels of these proteins in patient samples, which was considerably below the detection threshold for ELISA. In paper II, we developed and adapted a new method into the in situ PLA methods for detection and identification of extracellular vesicles (EVs) using flow cytometry as readout (a method we call ExoPLA). We identified five target proteins on the surface of the Evs and using three colors, we identified the EV using flow cytometer. In paper III, we aim to improve the efficiency of in situ PLA by creating and developing new designs and versions of the assay we called Unfold probes Through comparison of detection of protein using in situ PLA versus Unfold probes, we observed considerable decrease in non-specific signals, and also a lower detection threshold. In paper IV, we describe the development of a solid phase proximity extension (sp-PEA) assay for protein detection and quantification. We compared detection of IL-8, TNF-alpha, IL-10 and IL-6 using spPEA and PEA; spPEA demonstrations over 2 orders of magnitudes in the lower detection concentrations by decreased in background noise.
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Wai, Chi-wan, and 衛至韻. "Development of shell vial culture assay for the rapid diagnosis of respiratory viruses using the human colorectal adenocarcinoma (CaCo2) cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193551.
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Background: Respiratory diseases are common worldwide, which are caused by various respiratory viruses. As symptoms caused by these viruses are similar, laboratory diagnosis is essential to distinguish the virus. Conventionally, respiratory viruses are isolated by cell culture with a panel of cell lines. However, handling of several cell lines is labour intensive, and the turnaround time of conventional culture is long. In previous study, the use of human colon adeno-carcinoma (Caco-2) in conventional culture was investigated. The study has proven that Caco-2 is generally susceptible to the eight common respiratory viruses, i.e. Adenovirus, Influenza A and B, Respiratory Syncytial virus, Parainfluenza virus 1, 2,3 and 4. As turnaround time of conventional culture is long; therefore, in this study, rapid shell vial culture using Caco-2 cells were evaluated. Moreover, the application of Caco-2 shell vial culture on recovering human metapneumovirus (hMPV) was also investigated.
Materials and methods: This study consisted of four stages. First, recovery of viruses by conventional culture and shell vial culture of Caco-2 were compared. Specimens were added to conventional culture and shell vial simultaneously. For conventional culture, formation of CPE was examined daily and IF staining was performed when CPE was indicated; meanwhile, shell vial culture were incubated for seven days and stained with IF to detect infected cells. In stage two, the effect of incubating shell vial culture in rolling drum was investigated. Shell vials inoculated with the same specimen in duplicate were incubated in rolling drum and without rolling drum simultaneously. IF staining was performed in day 2, and results were obtained. For those which are IF negative in day 2, second shell vial was further incubated to seven days before harvest. In the next stage, a large batch of samples was used to evaluate on the use of Caco-2 shell vial culture in day 2 and day 7. Lastly, Caco-2 shell vial and conventional culture and LLC-MK2 conventional culture were tested for isolation of hMPV.
Results: Compared to Caco-2 conventional culture, recovery rate of shell vial culture was elevated slightly. When experimenting on the effect of incubation in rolling drum, results showed that recovery rate was raised in shell vial with rolling drum in day 2, moreover, the percentage of positive cells were increased significantly (p value < 0.05). Furthermore, in the evaluation of Caco-2 shell vial in day 2 and day 7, 75% of samples were isolated in day 2 while 85% were recovered in day 7. Lastly, in the investigation on recovery of hMPV, 53%, 42% and 17% hMPV positive cases were isolated by Caco-2 shell vial, Caco-2 conventional culture and LLC-MK2 conventional culture respectively.
Conclusion: First, although recovery rate by shell vial and conventional culture were similar, turnaround time was reduced from a week to a few days by shell vial culture. Therefore, Caco-2 shell vial culture is a more efficient than Caco-2 conventional culture in isolating respiratory viruses. The study also showed that incubation of shell vial in rolling drum able to increase the number of positive cells. Furthermore, in this study, Caco-2 cells were also shown to be more efficient in isolating hMPV when compare to LLC-MK2 cells.<br>published_or_final_version<br>Microbiology<br>Master<br>Master of Medical Sciences
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Mareledwane, Vuyokazi Epipodia. "Heterologous expression of African horsesickness virus VP2 and the development of a potential diagnostic assay." Diss., University of Pretoria, 2010. http://hdl.handle.net/2263/30991.
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Gokhale, Priyanka G. "DEVELOPMENT AND COMMERCIALIZATION OF A FECAL DNA BASED MOLECULAR DIAGNOSTIC ASSAY FOR COLORECTAL CANCER SCREENING." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1275440415.
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Black, Kelley Elizabeth. "Development of a multiplex bead assay to detect exposures to tick-borne diseases in dogs and a comparative performance analysis." Thesis, Kansas State University, 2016. http://hdl.handle.net/2097/32948.
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Master of Science<br>Department of Diagnostic Medicine/Pathobiology<br>Melinda J. Wilkerson<br>Tick-borne bacteria, Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis are significant zoonotic pathogens of dogs and humans worldwide. In tropical regions such as Grenada, West Indies, dogs represent a major reservoir for E. canis and A. platys, and they are often co-infected. The purpose of this study was to develop a serologic, multiplex bead-based assay to detect species-specific exposures to E. canis, A. platys, and E. chaffeensis in dogs for purposes of surveillance and public health. Peptides from specific outer membrane proteins of P30 for E. canis, OMP1X of A. platys, and P28-19/P28-14 of E. chaffeensis were coupled to magnetic beads and assays were optimized using the multiplex Luminex xMAP® platform. In experimentally infected dogs, the multiplex assay successfully detected antibodies for E. canis and E. chaffeensis, but not A. platys. In the Grenadian population (n=104), the multiplex assay and the in-house ELISA, the SNAP® 4Dx®, detected A. platys antibodies as well as Ehrlichia spp.. Multiplex assay results were found to have “good” and “very good” agreement with the ELISA and IFA for E. canis antibody-positive dogs (K value of 0.73 and 0.84 respectively), while ELISA and IFA had “very good” agreement with each other (K value of 0.85). A. platys multiplex results had only “poor” agreement with ELISA and IFA (K value of -0.02 and 0.01, respectively), while the ELISA and IFA tests had “moderate” agreement with each other (K value of 0.5). These tests showed the prevalence of exposure to E. canis to be comparable with previous studies (38% in 2014), but a doubling of exposure to A. platys determined by IFA and 4Dx® from 9% in 2006, to 20% in 2014. Bayesian modeling (performed on E. canis data only) suggested conditional independence between the IFA, 4Dx®, and MAG tests using consensus priors calculated from literature, and that the bead-assay had comparable sensitivity and specificity to the IFA and ELISA tests. In conclusion, the multiplex peptide assay performed well in detecting the seropositive status of dogs to E. canis and had good agreement with commercial assays; however, more work needs to be done to assess performance in populations of dogs with exposures to multiple species of Ehrlichia. Further, the reasons for low seroreactivity to A. platys need to be further investigated.
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Osborn, Rachel Kathleen. "Identification of microbes degrading nematicides and the development of a diagnostic assay for nematicide persistence in soils." Thesis, Open University, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424824.
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Botha, Elizabeth Magdelena. "Molecular characterization of South African lineage II West Nile virus isolates and development of a diagnostic assay." Diss., University of Pretoria, 2008. http://hdl.handle.net/2263/25469.
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West Nile virus (WNV) belongs to the Flaviviridae family, a virus family of which many members are known as human pathogens. WNV has a worldwide distribution and strains that cluster in lineage II is endemic to sub-Saharan Africa. The complete nucleotide sequence of four lineage II West Nile virus strains, isolated in South Africa from patients with mild or severe WNV infections, were determined. Using a murine model, these strains had been shown to produce either highly or less neuroinvasive infections and induced similar genes to corresponding highly or less neuroinvasive lineage I strains. Nucleotide and amino acid sequence comparison between highly and less pathogenic lineage II strains demonstrated that the non-structural genes and in particular the gene coding for the NS5 proteins were the most variable. All the lineage II strains sequenced in this study were found to possess the E-protein glycosylation site previously postulated to be associated with virulence. Comparison of the signalase cleavage sites suggested that lineage II strains may be cleaved slightly more efficiently than lineage I strains in the C-prM junction, but less efficiently between prM and E genes. Relative to the highly neuroinvasive strains sequenced in this study major deletions were found in the 3’ noncoding region of 2 lineage II strains shown in previous studies to be either less- or not at all neuroinvasive. This is the first report of full genome sequences of highly neuroinvasive lineage II WNV strains. Currently available commercial WNV ELISA kits were developed with lineage I WNV strains and are expensive to use. For these reasons the development of a potential ELISA diagnostic assay based on the South African lineage II strain, H442, was envisaged. Such assay, if reliable and efficacious would be a useful tool towards WNV surveillance. The prM and E genes were selected to be expressed as recombinant antigens because of their co-expression nature and because the envelope protein is the principal target for neutralization. After cloning of the respective genes and verification of integrity, a mammalian expression system was utilized. Different mammalian cells and transfection media were tested and BHK 21 cells with SuperFect transfection medium were found to be best. Attempted expression of proteins was tested with immunofluorescent antibody testing as well as SDS-PAGE and Western blot analysis. Expression of recombinant WNV antigens were also tested in indirect and sandwich ELISA’s systems. It was however not possible to perform these two ELISA systems at a satisfactory level or clearly indicated if expression of proteins was successful.<br>Dissertation (MSc (Microbiology))--University of Pretoria, 2008.<br>Microbiology and Plant Pathology<br>unrestricted
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Phillips, Mallory Elizabeth. "Epitope mapping of African swine fever virus p72 capsid protein using polyclonal swine sera and monoclonal antibodies." Thesis, Kansas State University, 2016. http://hdl.handle.net/2097/34528.
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Master of Science<br>Department of Diagnostic Medicine/Pathobiology<br>Raymond R. R. Rowland<br>African swine fever is a hemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV), a double-stranded DNA virus and the only member of the family Asfarviridae. The structure of this multilayer virion contains more than 34 proteins including the protein p72 which is the major capsid protein. A single conformational neutralizing epitope has been identified on p72, but information on the other antigenic regions (epitopes) is lacking. The objective of this study was to identify p72 epitopes using polyclonal swine sera and a panel of monoclonal antibodies with the ultimate goal being the development of a blocking ELISA assay for the detection of anti-ASFV antibodies. The segment of the p72 protein from amino acids 1 to 345 was divided into five overlapping fragments which were then commercially synthesized. These fragments were cloned into the pHUE expression vector and transformed into Escherichia coli competent cells. The recombinant proteins were expressed in vitro, purified, and used as antigens in indirect ELISAs and western blots to test monoclonal antibodies and polyclonal swine sera. The monoclonal antibodies were produced against the p72 protein based on the ASFV Georgia/07 strain. The polyclonal sera were obtained from pigs immunized with a defective alphavirus replicon particle, RP-sHA-p72, expressing a recombinant protein composed of the extracellular domain of the ASFV HA protein together with the whole p72 protein. The polyclonal sera reacted to p72 in two distinct regions: between amino acids 1 and 83 and between amino acids 250 and 280. The anti-p72 reactive monoclonal antibodies reacted with p72 in three regions: between amino acids 100 and 171, amino acids 180 and 250, and amino acids 280 and 345. Fine mapping with oligopeptides allowed for the identification of six different linear epitopes. Among the monoclonal antibodies selected for blocking assay development, two have been shown to be promising candidates for further evaluation using sera from ASFV-infected pigs.
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Ohlsson, Sandra. "Evaluation and development of reagents and improved protocol for flow cytometry readout using in situ PLA." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-166431.
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The diagnosis of cancer today is obsolete, depending upon pattern recognition and non-quantifiable data. The time consuming diagnosis is often performed on biopsies, fixed using non standardised procedures, and leaves room for dubious results. The diagnosis is also invasive, exposing patients to risk of infections and discomfort due to the need of tissue samples. The knowledge about changes in protein expression levels related to cancer can instead be utilized to generate a new diagnostic tool. By adapting the in situ proximity ligation assay (in situ PLA) to cells in solution, it is possible to detect proteins, or protein interactions, within cells without the need for tissue samples. Since the method is both highly sensitive and specific, it delivers reliable results. In this report, the in situ PLA method for cells in solution is combined with flow cytometry readout. Hence, a new and less invasive diagnostic tool for cancer, delivering highly accurate high throughput single cell analysis, may be on the rise.
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Morar, Darshana. "Development of ELISAs for the detection of interferon-gamma in rhinoceroses and elephants as diagnostic tools for Mycobacterium bovis and Mycobacterium tuberculosis infections." Thesis, Pretoria : [s.n.], 2009. http://upetd.up.ac.za/thesis/available/etd-12032009-193314/.
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Key, Kijona Farthing. "Molecular characterization of the major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) and evaluation of its use for a diagnostic assay, vaccine development, and the examination of quasispecies evolution." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27282.
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Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has devastated the global swine industry since the mid 1980s. Although modified live vaccines (MLVs) are typically used for the prevention of clinical disease, they are not always fully effective. Additionally, acute PRRS outbreaks, characterized by more severe clinical signs, have appeared in herds that were previously vaccinated. In this dissertation, we further analyzed the pathogenesis of PRRSV through genetic characterization, assay development, and quasispecies evaluation using the PRRSV ORF5 gene while also attempting to develop an improved PRRS vaccine.
To explore the possible mechanism for the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. Sequence and phylogenetic analyses revealed that seven of the acute PRRS virus (PRRSV) isolates were related to other N. American PRRSV isolates while one isolate, 98-37120-2, was very closely related to and may have been derived from the MLV, RespPRRS. We also developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV field isolates with significant nucleotide sequence identities (â d98%) with the MLVs based on the amplification, denaturation, and reannealing of the ORF5 gene of the field isolates with those of MLV reference strains. All of the field isolates that were highly related to RespPRRS (â T2% nucleotide sequence divergence) were identified by the HMA to form homoduplexes with the reference RespPRRS MLV.
We also developed a unique strategy for infecting pigs with PRRSV, known as in vivo transfection, by bypassing the traditional in vitro cell culture step required for in vivo studies. We demonstrated that inoculation of RNA transcripts of a PRRSV infectious cDNA clone directly into the lymph nodes and tonsils of pigs produces active PRRSV infection. Using this method, we also examined the quasispecies populations of PRRSV. Finally, we evaluated the ability of Salmonella choleraesuis to express the PRRSV GP5, and tested its immunogenicity in mice. Based on our data, there was no indication of Salmonella replication in the mice or any evidence of antibody production against S. choleraesuis or PRRSV GP5.<br>Ph. D.
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León, janampa Nancy. "Development of a test associated with magnetic nanoparticles for the diagnosis of tuberculosis." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0272.
Full textAbstract:
Mycobacterium tuberculosis provoque l'une des maladies qui présentent le taux de mortalité et de morbidité le plus élevé des Amériques et du monde. Dans les pays en développement, le diagnostic de la tuberculose (TB) repose sur la microscopie des frottis et des cultures bactériologiques. La première méthode a une faible sensibilité et la seconde met plusieurs semaines à atteindre un diagnostic de confirmation. L'absence de diagnostic rapide compromet les efforts de lutte contre la tuberculose, favorisant ainsi sa transmission à la population vulnérable. Actuellement, les nanoparticules magnétiques (MNP) fonctionnalisées par des biomolécules sont utilisées en biomédecine, en raison de leurs propriétés magnétiques, électriques et optiques. De cette manière, en appliquant des champs magnétiques externes, les MNP bio-fonctionnalisées sont utilisées pour détecter et concentrer les cellules et les biomolécules à partir d'échantillons biologiques.Dans ce travail, nous présentons la synthèse, la caractérisation et la bio-fonctionnalisation de nanoparticules magnétiques afin de développer un test ELISA en sandwich associé aux MNP, afin de détecter les antigènes de M. tuberculosis. À cette fin, des nanoparticules magnétiques ont été synthétisées par une méthode de co-précipitation. La surface des MNP a été amino-silanisée (MNP@Si@NH2) et caractérisée par des méthodes physico-chimiques.Les antigènes MTB évalués dans cette étude étaient: Hsp16.3, CFP10, ESAT6, MTC28, MPT64, protéine de 38 kDa, Ag85B et MoeX. Le clonage et l'expression de protéines recombinantes ont été réalisés dans le système de E. coli BL21 (DE3) pLysS. Des anticorps polyclonaux ont été produits chez des lapins Nouvelle-Zélande et des souris BALB/C, préalablement immunisés avec des antigènes recombinants purifiés. Des anticorps spécifiques (ab) ont été immobilisés sur les surfaces des MNP amino-silanisées. Les MNP@Si@ab ont été associées dans un test ELISA sandwich colorimétrique pour capturer et détecter les antigènes natifs du MTB à partir d’échantillons d’expectorations.La XRD, la spectroscopie Mössbauer, le potentiel zêta, la TEM et le FTIR ont validé l'obtention des MNP montrant un diamètre de cristal de diffraction de ~ 12,5 nm (10,48 ± 2,56 nm), une charge nette superficielle de +23,57 ± 2,87 mV, des profils caractéristiques de magnétite et une structure sphérique. De plus, une saturation en aimantation de 37,06 emu.g-1 a été observée. Pour la fonctionnalisation des surfaces de nanoparticules avec des anticorps, une méthode via l'utilisation d'ester activé (agent de couplage EDC/NHS) a été utilisée pour la formation de liaisons peptidiques. Des paramètres tels que le temps d'incubation, la concentration des agents de couplage et le niveau de saturation de surface des MNP amines silanisées (MNP@Si@NH2) ont déjà été standardisés.Enfin, des anticorps fonctionnalisés sur des MNP ont été utilisés pour capturer et détecter les antigènes recombinants et natifs de M. tuberculosis dans un test sandwich ELISA-MNP@Si@ab (dans un temps de réaction <4 h). Les antigènes ESAT6 et CFP10 ont été mieux différenciés dans les expectorations des patients atteints de tuberculose (fold value ~ 1,8). L'utilisation de MNP@Si@ab a amélioré la détection des antigènes du MTB dans des échantillons biologiques. Nos résultats sont encourageants, mais des évaluations supplémentaires sont nécessaire telles que la détermination de réactions croisées avec des échantillons d'expectorations provenant de patients atteints d'autres infections, la réalisation du test avec les expectorations fraîches de patients tuberculeux et la détermination de la sensibilité et de la spécificité de la méthode<br>Mycobacterium tuberculosis causes one of the diseases with the highest mortality and morbidity rate in the Americas and around the world. In developing countries, the diagnosis of tuberculosis (TB) is based on smear microscopy and bacteriological cultures. The first method has low sensitivity, and the second take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control TB, favoring its transmission to the susceptible population. Currently, magnetic nanoparticles (MNPs) functionalized with biomolecules have been used in biomedicine, due the magnetic, electrical and optical properties. In this way, applying external magnetic fields, bio-functionalized MNPs is used to detect and concentrate cells and biomolecules from biological samples.In this work we present the synthesis, characterization and bio-functionalization of magnetic nanoparticles, to develop a sandwich ELISA assay associated to MNPs to detect antigens from M. tuberculosis. For this purpose, magnetic nanoparticles were synthesized by co-precipitation method. The MNP surface was amine-silanized (MNP@Si@NH2) and characterized by physical-chemical methods.The MTB antigens evaluated in this study were: Hsp16.3, CFP10, ESAT6, MTC28, MPT64, 38 kDa protein, Ag85B and MoeX. Cloning ad expression of recombinant proteins were made in E. coli BL21 (DE3) pLysS system. Polyclonal antibodies were produced in New Zealand rabbits and BALB/C mice, previously immunized with purified recombinant antigens. Specific antibodies (ab) were immobilized in the amine-silanized MNP surfaces. The MNP@Si@ab were associated in a colorimetric sandwich ELISA assay to capture and detect native MTB antigens from sputum samples.The XRD, Mössbauer spectroscopy, zeta potential, TEM and FTIR demonstrated the successful preparation of the MNPs showing a diffraction crystal diameter of ~12.5 nm (10.48 ± 2.56 nm), superficial net charge of ᶎ: +23.57 ± 2.87 mV, characteristic patterns of magnetite and a spherical structure. Additionally, a magnetization saturation of 37.06 emu.g-1 was observed. For the functionalization of nanoparticle surfaces with antibodies, active ester method (coupling agent EDC/NHS) were used for peptide bond formation. Parameters such as time of incubation, concentration of coupling agents and surface saturation level of amine-silanized MNPs (MNP@Si@NH2), were previously standardized.Finally, antibody functionalized on MNPs were used to capture and detect recombinant and native M. tuberculosis antigens in an ELISA-MNP@Si@ab sandwich test (in a reaction time <4 h). The ESAT6 and CFP10 antigens were better discriminated in sputum pooles from patients with TB (fold value ~ 1.8). The use of MNP@Si@ab improved the detection of MTB antigens in biological samples. Our results are encouraging, but the essay requires additional evaluations such as determining cross-reactions with sputum samples from patients with other infections, performing the test with fresh sputum of TB patients, and determining the sensitivity and specificity of the method<br>Mycobacterium tuberculosis causa una de las enfermedades con la tasa más alta de mortalidad y morbilidad en las Américas y en todo el mundo. En países en vías de desarrollo, el diagnóstico de tuberculosis (TB) se basa en microscopía de frotis y cultivos bacteriológicos. El primer método tiene baja sensibilidad y el segundo toma varias semanas para llegar a un diagnóstico confirmatorio. La falta de un diagnóstico rápido compromete los esfuerzos para controlar la TB, lo que favorece su transmisión a la población susceptible. Actualmente, las nanopartículas magnéticas (MNP) funcionalizadas con biomoléculas se han utilizado en biomedicina, debido a las propiedades magnéticas, eléctricas y ópticas. De esta manera, aplicando campos magnéticos externos, se utilizan MNP bio-funcionalizadas para detectar y concentrar células y biomoléculas a partir de muestras biológicas. En este trabajo presentamos la síntesis, caracterización y bio-funcionalización de las nanopartículas magnéticas para desarrollar un ensayo ELISA sándwich usando MNPs para detectar antígenos de M. tuberculosis. Para este propósito, las nanopartículas magnéticas fueron sintetizadas por el método de co-precipitación. La superficie de MNP fue amino-silanizada (MNP@Si@NH2) y se caracterizada por métodos físico y químicos. Los antígenos de MTB evaluados en este estudio fueron: Hsp16.3, CFP10, ESAT6, MTC28, MPT64, proteína de 38 kDa, Ag85B y MoeX. La clonación y la expresión de las proteínas recombinantes se realizaron en el sistema de E. coli BL21 (DE3) pLysS. Se produjeron anticuerpos policlonales en conejos de Nueva Zelanda y ratones BALB/C, inmunizados previamente con antígenos recombinantes purificados. Se inmovilizaron anticuerpos específicos (ab) en las superficies de MNP amino-silanizadas (MNP@Si@ab). El MNP@Si@ab fue utilizado en un ensayo ELISA sándwich colorimétrico para capturar y detectar antígenos de MTB nativos en muestras de esputo. La XRD, espectroscopia de Mössbauer, la potencial zeta, TEM y FTIR demostraron la preparación exitosa de los MNP, el cual mostró un diámetro de difracción del cristal de ~ 12.5 nm (10.48 ± 2.56 nm), carga neta superficial de ᶎ: +23.57 ± 2.87 mV, patrones característicos de magnetita y una estructura esférica. Además, una saturación de magnetización de 37.06 emu.g-1 fue observada. Para la funcionalización de superficies de nanopartículas con anticuerpos, se utilizó el método del éster activo para la formación de enlaces peptídicos. Parámetros tales como el tiempo de incubación, la concentración de los agentes de acoplamiento y el nivel de saturación de la superficie de las MNPs aminosilanizadas (MNP@Si@NH2) fueron estandarizadas. Finalmente, se usaron MNP funcionalizados con anticuerpos para capturar y detectar antígenos nativos y recombinantes de M. tuberculosis en una prueba sándwich de ELISA-MNP@Si@ab en un tiempo de reacción <4 h. Los antígenos ESAT6 y CFP10 se discriminaron mejor en las muestras de esputo de los pacientes con TB (fold value ~ 1,8). El uso de MNP@Si@ab mejoró la detección de antígenos de MTB en muestras biológicas con respecto a un sELISA convencional. Nuestros resultados son alentadores, pero el ensayo requiere evaluaciones adicionales, como determinar reacciones cruzadas con muestras de esputo de pacientes con otras infecciones, realizar la prueba con esputo frescos de pacientes con TB y determinar la sensibilidad y especificidad clînica del método
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Wong, Ka-lun, and 王嘉倫. "Development of loop-mediated isothermal amplification assay for rapid diagnosis of tuberculosis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193531.
Full textAbstract:
Tuberculosis (TB), a severe disease caused by Mycobacteria tuberculosis (MTb), remains a globally severe health problem. In modern days, emergence of drug resistant TB is a new threat to public health since it can lead to treatment failure, increased transmission of TB to other hosts and further development of drug resistant complications. The traditional diagnostic method for TB by Acid Fast Bacilli (AFB) smear and Löwenstein–Jensen medium (LJ) culture are poor in sensitivity and time consuming respectively. There is a need for rapid diagnosis and identification of MTb. Nucleic acid amplification like PCR and real-time PCR are options for rapid diagnosis. However, such techniques require sophisticated technique and complex equipment. The high cost would constitute a barrier for countries with a high demand but only limited resources. Loop-mediated isothermal amplification (LAMP) assay is a novel technique, proven by many studies as to its high sensitivity and highly specificity to MTb. Most importantly, LAMP assay is economical and affordable by developing countries.
The first objective of this study was to evaluate the analytical sensitivity and specificity of LAMP assay. The specificity of LAMP assay was determined by performing LAMP assay on 19 clinical isolates, which had already been identified previously. The clinical isolates included 14 mycobacteria tuberculosis complex (MTb), and five mycobacteria other than tuberculosis (MOTT) strains that were positive for AFB smear and LJ culture but negative for IS6110 single-tube nested real time PCR. The specificity was 100%. The analytical sensitivity as well as the limit of detection (LOD) were determined by testing on a duplicate set of serial DNA dilution, where each duplicate consisted of dilution of 100,000, 10,000, 1000, 300, 100, 10 and 1 colony forming unit/milliliter (CFU/ml). The LOD of LAMP assay was about 3 CFU per reaction.
The second objective of this study evaluated the diagnostic performance of LAMP assay against AFB culture and IS6110 single tube nested real time PCR for identification of MTb in 200 respiratory specimens from 123 patients. All the specimens have already been tested for IS6110 single tube nested real time PCR, and culture results and AFB smears results have been obtained for all the specimens. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval using LJ culture as gold standard. The LAMP assay had a sensitivity of 80%, specificity of 92.6%, PPV of 60% and NPV of 97.1%. However, MTb might fail to grow on the LJ culture medium for various reasons, for instance, MTb might already be dead after antibiotic treatment of the patient, or there might be poor laboratory practices during the processing of the specimens. Since the LJ culture method could produce false negatives in the situations described above, an alternative to the LJ culture method, ‘Hybrid Method’ was used as the gold standard. Under this method, a specimen was regarded as positive if the LJ culture result was positive. On the other hand, if a specimen generated a negative result using the LJ culture method, the results from the LAMP assay and IS6110 nested real time PCR would be considered, i.e., if both the LAMP assay and IS6110 nested real time PCR gave positive results while the results of LJ culture were negative, the specimen was referred to be positive in this case. In other words, a specimen would be regarded as negative if and only if the LJ culture result was negative and at least one of the LAMP assay or IS6110 was negative at the same time. Along the same line, the sensitivity, specificity, PPV and NPV of the three diagnostic methods (AFB smear microscopy, LAMP assay amplification and IS6110 single-tube nested real time PCR) were calculated with 95% confidence interval against the Hybrid Method. After resolution, the LAMP assay had a sensitivity of 87.0%, specificity of 100%, PPV of 100% and NPV of 97.1%.
Our results showed that the LAMP assay has a great potential to be a new TB diagnostic test, especially in developing countries, with its lot of advantages like ease of use, cheap and fast. The LAMP assay in the study showed a high specificity, however, the sensitivity has to be improved before application in clinical use. For comparison of clinical performance, IS6110 single tube nested real time PCR had a higher sensitivity than that of LAMP assay (100% vs 80% using culture as gold standard; 100% vs 87% using ‘Hybrid Method’ as gold standard). However, LAMP assay had a higher specificity than that of IS6110 single tube nested real time PCR (92.6% vs 90.7% using culture as gold standard; 100% vs 98% using ‘Hybrid Method’ as gold standard). LAMP had been proven to be a potential and powerful tool in clinical diagnosis of MTb. Further improvement on its sensitivity is required to enable its extensive use in the clinic in the future.<br>published_or_final_version<br>Medical Sciences<br>Master<br>Master of Medical Sciences
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Fluegel, Amanda M. "Validation of diagnostic assays and development of molecular epidemiological tools for brucellosis." Laramie, Wyo. : University of Wyoming, 2008. http://proquest.umi.com/pqdweb?did=1594477821&sid=1&Fmt=2&clientId=18949&RQT=309&VName=PQD.
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Murphy, Joseph Francis. "The development of enzyme-linked immunosorbent assays (ELISA) for the catecholamines." Thesis, University of Salford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292901.
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Löf, Liza. "Applications of in situ proximity ligation assays for cancer research and diagnostics." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-300191.
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In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform. In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow. Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma. In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system. Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt. A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry.
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Foley, Jennifer Olivia. "Design and development of surface plasmon resonance imaging microfluidic assays /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/7982.
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Ng, Dione K. M. "Development of a Multiplex Exoglycosidase Assay for Diagnosis of Oligosaccharidoses using Tandem Mass Spectrometry." Thesis, University of Ottawa (Canada), 2011. http://hdl.handle.net/10393/28924.
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Oligosaccharidoses are Lysosomal Storage Disorders (LSDs) that result from mutations in genes encoding exoglycosidases, leading to accumulation of unmetabolized N-linked oligosaccharides within lysosomes. Age at onset and rate of disease progression vary among patients. Diagnosis based solely on clinical presentation is often challenging because of overlapping clinical symptoms between these disorders. The aim of this research is to use tandem mass spectrometry (MS/MS) to establish a multiplex method to measure exoglycosidase activities, in dried blood spots (DBS), involved in the degradation of N-linked oligosaccharides using natural substrates. Current fluorometric assays for each exoglycosidase using specific 4-methylumbelliferyl (4MU) substrates allow enzyme activities to be determined separately from a variety of human tissue sample types. A universal buffer was established by comparing these assay conditions to allow multiplexing of the exoglycosidases in a single vial. Initial attempts to develop an enzyme activity assay using disaccharides as the starting substrate and by monitoring unique monosaccharide products by MS/MS after exposure to an enzyme source from cultured skin fibroblasts were unsuccessful due to interfering endogenous hexose isomers. Taking another approach, multiplexing was successfully demonstrated for beta-Galactosidase and beta-Hexosaminidase using alternative substrates. 4MU and paranitrophenol (PNP) conjugated to particular monosaccharides allowed 4MU and PNP products to be measured and enzyme activities to be calculated. Here, we provide a proof of principle that MS/MS technology can allow simultaneous multiplexing of several enzyme activities using distinctive starting substrates. A multiplex assay for the remaining exoglycosidases can still permit the development of an Oligosaccharidoses screening test to assist clinical diagnosis.
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De, Wit Deo. "The development of a polymerase chain reaction assay for the detection of non-tuberculous mycobacteria." Master's thesis, University of Cape Town, 1990. http://hdl.handle.net/11427/25667.
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Wu, Liang-Ta. "Development and evaluation of influenza molecular diagnostic assays intended for point-of-care testing." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607758.
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Ruiz, Vega Gisela. "One-step electrochemical magneto assays for the development of point-of-care (POC) diagnostic devices." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/669860.
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Un dels majors reptes per a monitoritzar i millorar la salut de la població a nivell mundial és la manca de proves de diagnòstic apropiades per a la detecció primerenca de malalties, la selecció de tractaments apropiats i el seguiment de pacients al llarg del temps. La disponibilitat d’eines de diagnòstic prou ràpides, sensibles i robustes és crucial per aconseguir el benestar dels pacients a tot el món. En aquest context, la nanotecnologia i el desenvolupament de biosensors són camps en ràpida evolució que han generat grans expectatives, produint proves més ràpides i més fàcils de realitzar que la majoria dels mètodes clàssics. Els biosensors s’han descrit en base a l’ús d’una àmplia varietat d’elements de biotecnologia i tipus de transducció de senyals. Entre ells, els biosensors electroquímics són el tipus més comú en ús avui en dia gràcies a la portabilitat i el baix cost de l’equip de mesura, les mesures ràpides, robustes i quantitatives proporcionades, i la facilitat de miniaturització de tot el sistema de detecció. La recent incorporació de paper per a la producció d’elèctrodes impresos en paper i assajos electroquímics de flux lateral està fomentant el desenvolupament de dispositius extremadament econòmics que, gràcies a les propietats fluídicas del paper, permeten reduir la complexitat de l’assaig i el nivell de manipulació per al usuari final. Això afavoreix el desenvolupament de dispositius de diagnòstic de \”Point-of-care\” (POC), que poden ser utilitzats directament pel pacient o als centres d’atenció primària de salut. D’altra banda, les partícules magnètiques (PM) s’han utilitzat amb gran èxit en l’optimització dels magneto-biosensors. Les PM són atractives per a aquest propòsit perquè, un cop modificades amb un bioreceptor apropiat, atorguen una preconcentració simple, ràpida i específica de l’analit objectiu. Les PM també ofereixen superfícies actives en 3D relativament grans, que es barregen amb agitació constant amb les mostres i permeten una ràpida unió amb els analits. No obstant això, les PM també presenta limitacions, com el seu maneig tediós i lent que només està a l’abast d’usuaris altament capacitats. L’objectiu principal d’aquest projecte de tesi doctoral va ser la producció de magneto-biosensors electroquímics ràpids, fàcils de realitzar, robustos i sensibles per a la detecció de biomarcadors de diagnòstic en mostres de sèrum, plasma i sang. Com es mostrarà, això s’ha aconseguit en dos nivells. Primer, desenvolupant un format de magneto-immunoassaig extremadament ràpid i simple. En segon lloc, fabricant elèctrodes de paper microfluids simples i econòmics, que van ser explotats per dur a terme en el xip la majoria dels passos del magneto-immunoassaig simplificat amb la mínima intervenció de l’usuari.<br>Uno de los mayores desafíos para monitorear y mejorar la salud de la población a nivel mundial es la falta de pruebas de diagnóstico apropiadas para la detección temprana de enfermedades, la selección de tratamientos apropiados y el seguimiento de pacientes a lo largo del tiempo. La disponibilidad de herramientas de diagnóstico suficientemente rápidas, sensibles y robustas es crucial para lograr el bienestar de los pacientes en todo el mundo. En este contexto, la nanotecnología y el desarrollo de biosensores son campos en rápida evolución que han generado grandes expectativas, produciendo pruebas más rápidas y más fáciles de realizar que la mayoría de los métodos clásicos. Los biosensores se han descrito en base al uso de una amplia variedad de elementos de biotecnología y tipos de transducción de señales. Entre ellos, los biosensores electroquímicos son el tipo más común en uso hoy en día gracias a la portabilidad y el bajo costo del equipo de medición, las medidas rápidas, robustas y cuantitativas proporcionadas, y la facilidad de miniaturización de todo el sistema de detección. La reciente incorporación de papel para la producción de electrodos impresos en papel y ensayos electroquímicos de flujo lateral está fomentando el desarrollo de dispositivos extremadamente económicos que, gracias a las propiedades fluídicas del papel, permiten reducir la complejidad del ensayo y el nivel de manipulación para el usuario final. Esto favorece el desarrollo de dispositivos de diagnóstico de \”Point-of-care\” (POC), que pueden ser utilizados directamente por el paciente o en los centros de atención primaria de salud. Por otro lado, las partículas magnéticas (PM) se han utilizado con gran éxito en la optimización de los magneto-biosensores. Las PM son atractivos para este propósito porque, una vez modificados con un bioreceptor apropiado, otorgan una preconcentración simple, rápida y específica del analito objetivo. Las PM también ofrecen superficies activas en 3D relativamente grandes, que se mezclan bajo agitación constante con las muestras y permiten una rápida unión con los analitos. Sin embargo, las PM también presenta limitaciones, como su manejo tedioso y lento que solo está al alcance de usuarios altamente capacitados. El objetivo principal de este proyecto de tesis doctoral fue la producción de magneto-biosensores electroquímicos rápidos, fáciles de realizar, robustos y sensibles para la detección de biomarcadores de diagnóstico en muestras de suero, plasma y sangre. Como se mostrará, esto se ha logrado en dos niveles. Primero, desarrollando un formato de magneto-inmunoensayo extremadamente rápido y simple. En segundo lugar, fabricando electrodos de papel microfluidos simples y económicos, que fueron explotados para llevar a cabo en el chip la mayoría de los pasos del magneto-inmunoensayo simplificado con la mínima intervención del usuario.<br>One of the greatest challenges for monitoring and improving the health of the
population at a global level is the lack of appropriate diagnostic tests for early detection
of diseases, selection of appropriate treatments and patient follow-up over time. The
availability of sufficiently fast, sensitive and robust diagnostic tools will be crucial to
achieve patients’ well-being worldwide. In this context, nanotechnology and biosensor
development are rapidly evolving fields that have generated great expectations,
producing tests faster and easier to carry out than most classical methods. Biosensors
have been described based on the use of a wide variety of biotechnology elements and
types of signal transduction. Among them, electrochemical biosensors are the most
common type in use today thanks to the portability and low cost of the measuring
equipment, fast, robust and quantitative measures provided, and easiness of
miniaturization of the whole detection system. The recent incorporation of paper and
paper-like materials for the production of paper printed electrodes and lateral flow
electrochemical assays is fostering the development of extremely inexpensive devices
that, thanks to the fluidic properties of paper, allow reducing assay complexity and level
of manipulation for the end user. This favours the development of "Point-of-Care"
diagnostic devices (POC), which can be used directly by the patient or at primary health
care centres.
On the other hand, magnetic beads (MB) have been used with great success in the
optimization of magneto-biosensors. MB are attractive for this purpose because, once
modified with an appropriate bioreceptor, they grant simple, rapid and specific
preconcentration of the targeted analyte. MB offer also relatively large 3D active
surfaces, which mixed under constant agitation with the sample supply efficient and fast
analyte binding as well. Nevertheless, MB display limitations too, requiring tedious and
time-consuming handling that is only at reach of highly trained users.
The main objective of this PhD Thesis project was the production of rapid, easy to
perform, robust and sensitive electrochemical magneto-biosensors for the detection of
diagnostic biomarkers in serum, plasma and blood samples. As it will be shown, this has
been achieved at two levels. First, by developing an extremely fast and simple magnetoimmunoassay
format. Second, by fabricating simple and inexpensive microfluidic paper
electrodes, which were exploited to carry out on-chip most of the steps of the simplified
magneto-immunoassay with minimal user intervention.
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Bastos, Paulo André Dias. "Development of multiple reaction monitoring assays for bladder cancer diagnosis from urine samples." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22510.
Full textAbstract:
Mestrado em Bioquímica<br>O Carcinoma da Bexiga é uma doença maligna com extremas implicações
físicas e psicológicas para os pacientes e de elevadas repercussões socioeconómicas. A
falta de procedimentos de diagnóstico precoce não-invasivos tem permitido que a
sobrevivência destes pacientes tenha permanecido inalterada nos últimos 30 anos.
Desta forma, biomarcadores para diagnóstico não-invasivo são urgentemente
necessários, e amostras de urina representam o meio mais promissor para alcançar este
fim. Contudo, apesar de várias tentativas, ensaios imunológicos realizados em amostras
de urina demonstram fraca performance clínica e analítica.
Single/Multiple Reaction Monitoring (SRM/MRM) é uma técnica de espectrometria de
massa para quantificação exata e absoluta. SRM/MRM representa a alternativa mais
promissora para efeitos de quantificação, sendo altamente reprodutível, sensível e
robusta. Desta forma, objetivou-se o desenvolvimento de ensaios por SRM/MRM para
quantificação de biomarcadores de cancro da bexiga na urina, combinando múltiplos
marcadores num classificador unificador.
O ensaio MRM desenvolvido demonstrou em exatidão e especificidade equiparável ou
superior aos ensaios imunológicos até á data disponível. Combinando SLIT2, PROF1,
SPRC e NMP22 num classificador baseado em 4 marcadores resultou em performance
clínica comparável (~70% sensibilidade e ~100% especificidade ou ~80% sensibilidade e
~57% especificidade) quando comparado com os ensaios convencionais. Contudo, a
quantificação livre de interferências não pode ser assegurada devido a efeitos da matriz.
Um método eficiente e reprodutível para remover substâncias contaminantes presentes
na urina sem comprometer a deteção dos marcadores em causa é necessária para
atenuar os efeitos de matriz.<br>Bladder cancer is a malignant disease with extreme physical and psychological implications for the patients together with major economic societal costs.
The lack of early non-‐invasive diagnostic procedures has allowed survival outcomes to remain unaltered for the past 30 years.
Accordingly, non-‐invasive diagnostic biomarkers are urgently needed, and
urine samples represent the most promising means for non-‐invasive bladder cancer
diagnosis.
However, despite several encouraging claims, available immuno-‐based molecular assays display poor analytical and clinical performance in urine samples.
Single/Multiple Reaction Monitoring (SRM/MRM) is a high-‐performance mass spectrometry scanning mode for precise targeted quantification.
SRM/MRM represents the most promising approach for biomarker quantification purposes, as it is highly reproducible, sensitive and robust.
The main aim of this thesis was thus to develop a SRM/MRM-‐based assay for bladder cancer urinary biomarker quantification, combining multiple markers into a unifying classifier.
In addition, two independent chapters have been dedicated to i) the value of urine proteomics for disease diagnostics and to ii) the burden of the disease together with available tools for its diagnosis in the form of a literature meta-‐analysis and book chapter, respectively.
At the individual biomarker level, the MRM assay herein developed for urine profiling provided comparable-‐to-‐superior accuracy and specificity as comparedwhen to ELISA assays.
The combination of SLIT2, PROF1, SPRC and NMP22 in a 4-‐marker classifier resulted
in comparable-‐to-‐superior clinical performance (~70% sensitivity with ~100% specificity ~80% sensitivity with ~57% specificity) over conventional immuno-‐based assays.
However, interference-‐free measurements still could not be assured due to urinary
matrix effects.
A cost-‐efficient and reproducible method for the removal of unidentified urinary
contaminating substances without compromising the signal for the sought biomarkers
is required in order to counteract urinary matrix effects.
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Lewis, Sally. "Development of a Real-time Pcr Assay for the Detection of Campylobacter Jejuni and Campylobacter Coli." Thesis, University of North Texas, 2009. https://digital.library.unt.edu/ark:/67531/metadc9840/.
Full textAbstract:
Campylobacter organisms are the most commonly reported bacterial causes of foodborne infection in the world, with Campylobacter jejuni and Campylobacter coli responsible for over 99% of reported infections. Traditionally, Campylobacter species detection is an arduous process, requiring a special incubation environment as well as specific growth media for an extended growth period. The development of a rapid and reliable diagnostic tool for the detection of Campylobacter species would be a valuable aid to the medical diagnostic decision process, especially to rule out Campylobacter infection during the enteric pre-surgical time period. Improved patient outcomes would result if this rapid assay could reduce the number of enteric surgeries. Assays performed during this dissertation project have demonstrated that both SYBR® green and hydrolysis probe assays targeting an 84 nucleotide portion of cadF, a fibronectin-binding gene of Campylobacter jejuni and Campylobacter coli, were able to detect from 101 to 108 copies of organism from stool specimens, did not detect nonspecific targets, and exhibited a coefficient of variation (CV) of 1.1% or less. Analytical validation of sensitivity, specificity and precision, successfully performed in these studies, warrants additional clinical validation of these assays.
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Ismail, Kurimun. "Development and utilization of Luminex biomarker assays for diagnosis and monitoring of neurodegenerative disease." Thesis, Lancaster University, 2016. http://eprints.lancs.ac.uk/82998/.
Full textAbstract:
A common pathological feature of various neurodegenerative disorders is the accumulation of misfolded proteins in the brain. Neurodegenerative disorders associated with protein misfolding include Alzheimer’s disease (AD), Parkinson’s disease (PD), dementia with Lewy bodies (DLB), fronto-temporal lobar degeneration (FTLD), motor neuron disease (MND), Huntington’s disease and the prion diseases. The incidence and prevalence of most of these diseases is rising, especially those that cause dementia, due to an increase in the average human life span. The diagnosis of neurodegenerative disorders is heavily reliant on physical examinations and assessment of clinical symptoms. The clinical symptoms of many of these neurodegenerative diseases overlap, which poses a huge difficulty for accurate diagnosis, especially in the early stages. This has led to an interest in identifying reliable and robust discriminatory molecular biomarkers. A successful biomarker test will not only provide a more accurate means of diagnosis, but will allow efficient tracking of disease progression, benefitting the process of developing therapeutic strategies. In this project, the development and validation of a bead based assay system that has multiplexing capabilities (simultaneously measure multiple analytes in a single sample via a single assay) has been described. This assay system uses the Luminex technology and has been developed to quantitatively measure phosphorylated α-synuclein, total α-synuclein, total DJ-1 and LRRK2 in human CSF and plasma. These proteins are predominantly implicated in diseases collectively termed α-synucleinopathies. The initial aim of the project was to develop assays for proteins that span a range of neurodegenerative disorders, however, for reasons discussed in the final chapter of this thesis, this was not possible. This project provides evidence on how the use of plasma as a possible matrix for potential markers associated with brain diseases can be justified, since levels of phosphorylated α-synuclein in matched plasma and CSF samples positively correlated with each other. Plasma would be an ideal sample source for biomarker studies, since it is less invasive than obtaining CSF, thus allowing longitudinal studies to be performed. It was also shown how the DJ-1 protein in plasma may carry diagnostic potential by allowing differentiation between PD patients and healthy controls (p=0.004) as well as between PD and MSA patients (p=0.005). The discrimination between PD and MSA is vital since the two diseases are symptomatically very similar, thus posing a greater issue with accurate diagnosis. There has been minimal research discussing the presence of LRRK2 in human biological fluids such as plasma and CSF. This thesis presents the use of western blotting, high performance liquid chromatography (HPLC) and the Luminex technology as a means of detecting this protein in human CSF and plasma. The data related to LRRK2 in this thesis, opens up avenues for further research into this protein; to definitively show whether it can be detected in such biological fluids and whether it has any value as a biomarker.
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Monfort, James Daniel. "Optimization of culture-based diagnosis of campylobacter jejuni infections and development of a fecal assay for flagellar antigen /." The Ohio State University, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487685204970466.
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Merritt, Joshua. "Development and application of highly-parallel yeast functional assays for the analysis of mutant human proteins." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 1.77 Mb., 191 p, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3200528.
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Brown, Benjamin R. B. "Development of digital PCR DNA methylation assays for blood plasma-based diagnosis of lung cancer." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3021925/.
Full textAbstract:
Lung cancer is the leading cause of cancer-related death and is usually diagnosed at advanced stage leading to poor patient survival. Therefore there is a pressing need for early detection of disease. DNA methylation is an early event in carcinogenesis and a limited number of diagnostic markers have been developed for clinical use. This thesis seeks to address whether the development and application of novel DNA methylation assays can diagnose lung cancer at early stage. Previously identified DNA methylation biomarkers, along with novel targets identified by methylation microarray, were developed in multiplex assay format. Twelve markers were used to screen 417 bronchoalveolar lavage specimens from Liverpool Lung Project (LLP) subjects divided into training and validation sets. The optimal biomarker panel (CDKN2A, RARB and TERT) demonstrated improved clinical sensitivity and specificity (Sensitivity/Specificity: 85.7%/93.8%, AUC: 0.91) compared to previous studies. The optimal methylation algorithm detected more than 60% of stage T1 tumours and 93 cytologically occult lung cancer cases. Eight methylated DNA assays were optimised for use with the newly developed Droplet DigitalTM PCR (ddPCR) platform and a targeted pre-amplification technique, MethPlex enrichment, was developed. I established a comprehensive analytical framework to compare performance of methylation-specific ddPCR and quantitative methylation-specific PCR directly and in combination with MethPlex enrichment. ddPCR demonstrated greater precision and linearity, lower limit of detection (WT1 MethPlex ddPCR LOD95 = 1.86 GE), and discriminated twofold differences in methylated DNA input. MethPlex ddPCR detected DNA methylation more frequently in lung cancer patient plasma than in controls in a retrospective case-control study. Technical methylation controls were consistently and precisely detected at inputs as low as 3 methylated copies. Discriminatory efficiency of marker combinations was inadequate, presumably due to limitations in DNA extraction methodology. DNA methylation biomarker diagnostic performance in bronchoalveolar lavage merits further validation in a prospective trial. MethPlex ddPCR analysis showed great promise, demonstrating highly sensitive DNA methylation detection in technical assessment. It is expected that appropriate DNA extraction procedures and higher cfDNA yields will lead to much improved clinical discriminatory efficiency.
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Nyarku, Rejoice E. "Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer." Diss., University of Pretoria, 2020. http://hdl.handle.net/2263/77428.
Full textAbstract:
Brucellosis is an economically important bacterial disease of both animals and humans. In sub-Saharan Africa, the diagnosis of the disease remains a challenge. Brucellosis is underreported in South Africa, due to inconsistency in reports of bacteriological and serological tests, which lack adequate sensitivity and specificity in the diagnosis of the disease. They also are ineffective in confirming brucellosis during early stages of the disease.
The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid (rDNA) internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for early diagnosis of brucellosis and as a rapid screening tool. To achieve this, blood, milk and tissue samples were spiked with B. abortus biovar (bv.) 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The efficiency was 105% in tissue, 99% in blood, and 93% in milk. The 95% limit of detection (LOD) of the ITS qPCR assay was highest in tissue, followed by blood, then milk; thus (1.45, 13.30 and 45.54 bacterial genome copies/PCR reaction).
Furthermore, the diagnostic performance of the assay was compared to the Brucella cell surface protein real time polymerase chain reaction (BCSP31 qPCR) assay. Out of 56 aborted foetal tissue samples from bovine, ovine and caprine, 33% (19/56) were positive for Brucella spp. The sensitivity and specificity of the ITS qPCR assay were 87% and 95% respectively, compared to the 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The ITS qPCR gave earlier CT’s with a difference in CT (ΔCT) between ITS and BCSP31 ranging between 7.1 and 3.24.
The assay was efficient, sensitive and specific. It detected as little as 1.45 bacterial genome copies/PCR reaction in tissue, making this assay a valuable tool in early detection of the presence of the Brucella pathogen. It is sensitive and specific in the diagnosis of brucellosis.<br>Dissertation (MSc)--University of Pretoria, 2019.<br>Veterinary Tropical Diseases<br>MSc<br>Unrestricted
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Nicolini, Ariana Marie, and Ariana Marie Nicolini. "Single-Step, Optical Biosensors for the Rapid and Sensitive Detection of Bacterial and Viral Pathogens." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/623158.
Full textAbstract:
This dissertation discusses the development of inexpensive, easy-to-use, and field-deployable diagnostic techniques and devices for the early detection of various pathogens, commonly found in clinical samples and contaminated food and water. Infectious diseases account for about 90% of world health problems, killing approximately 14 million people annually, the majority of which reside in developing countries. In 2012, the World Health Organization (WHO) published data on the top 10 causes of death across the globe. Although communicable disease is a prevalent cause of fatality, both low-income and high-income countries, pathogen species and transmission are very different. Nearly 60% of deaths in developing countries are caused by food, water, air or blood-borne pathogens. The most prevalent illnesses are diarrheal disease, malaria, and HIV/AIDS. By contrast, the leading causes of death in developed countries (heart disease, cancer, and stroke) are not communicable and are often preventable. However, there is an increasing need for the development of rapid and accurate methods for pathogen identification in clinical samples, due to the growing prevalence of antibiotic-resistant strains. Incorrect, or unneeded antibiotic therapies result in the evolution of extremely aggressive nosocomial (hospital-acquired) infections, such as methicillin- (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The implementation of rapid, easy to use and cost-effective diagnostics will reduce the frequency of pathogen-related deaths in underdeveloped countries, and improve targeted antibiotic treatment in hospital settings, thus decreasing the potential development of more treatment-resistant "super bugs". This research includes novel techniques utilizing two major sensing modalities: serological (i.e. immunological), and nucleic acid amplification testing (NAATs). We first developed a highly sensitive (limit-of-detection = 100 CFU mL-1) particle immunoassay that takes advantage of elastic and inelastic light scatter phenomena, for optical detection of target antigens. This assay is performed upon a unique nanofibrous substrate that promotes multiplexing on a user-friendly platform. We then developed a novel technique, termed emulsion loop-mediated isothermal amplification (eLAMP), in which the target amplicon is detected in real-time, again utilizing light scattering detection and quantification. Both techniques require no sample pre-treatments, and can be combined with smartphone imaging for detection of targets in under 15 minutes. These methods have the potential to improve the speed and sensitivity of early pathogenic identification, thus leading to a reduction in preventative deaths and a decrease in global economic costs associated with infectious disease in clinical and other settings.
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Azmi, Suleiman Kifaya [Verfasser]. "Development and application of molecular diagnostic assays in the epidemiology of cutaneous leishmaniasis in the Jenin District / Kifaya Azmi Suleiman." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2014. http://d-nb.info/1046563661/34.
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Jäckel, Susanne [Verfasser]. "Development of novel diagnostic assays for the detection and surveillance of Rift Valley fever virus infections in ruminants and camels / Susanne Jäckel." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2015. http://d-nb.info/1073850544/34.
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Dernfalk, Johanna. "Multiplex flow cytometric assays for markers of inflammation : development and application in bovine samples /." Uppsala : Dept. of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, 2008. http://epsilon.slu.se/20085.pdf.
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Camarão, António Alexandre Riachos. "Development and optimisation of a group-specific real-time RT-PCR assay for the broad detection of the Simbu serogroup orthobunyaviruses." Master's thesis, Universidade de Lisboa, Faculdade de Medicina Veterinária, 2018. http://hdl.handle.net/10400.5/15823.
Full textAbstract:
The Simbu serogroup within the genus Orthobunyavirus belongs to the family Peribunyaviridae and comprises 32 recognised three-segmented negative-sense single-stranded RNA viruses, divided into two phylogenetic clades. Some members of this group of arthropod-borne viruses, cosmopolitan distributed, cause neurologic disease in humans as well as reproductive and neurologic disease in domestic animals, however, definitive diagnosis always requires laboratorial confirmation. Few real-time RT-PCR assays have been developed for the molecular diagnosis of Simbu serogroup orthobunyaviruses. There are two published methods with broad detection capacity, utilising either a SYBR Green based chemistry able to recognise viruses from both clades, which is not absolutely specific, or a TaqMan based chemistry that recognises only clade B viruses. A novel group-specific TaqMan-based real-time RT-PCR assay was developed, optimised and laboratory validated for the broad detection of the Simbu serogroup orthobunyaviruses. The published genomic data of the Simbu serogroup members were evaluated, and a conserved region, situated in the segment encoding the nucleocapsid protein, was selected to design a universal primer set and a pair of differently labelled hydrolysis probes, which allowed for the distinction between the two phylogenetic clades of the Simbu serogroup. Seven prototype Simbu serogroup isolates were used for the development of the assay, namely Akabane orthobunyavirus, Simbu orthobunyavirus, Shuni orthobunyavirus, Sathuperi orthobunyavirus, Shamonda orthobunyavirus, Ingwavuma virus and Sabo virus. The primer and probe concentrations in the reaction were optimised. Amplification efficiency was determined for each one of the viruses: AKAV (99%), SIMV (96%), SHUV (96%), SATV (97%), SHAV (84%), INGV (93%) and SABOV (110%). A panel constituted of genetically related, causative agents of abortion in ruminants and arthropod-borne viruses was selected for in vitro specificity analysis, and in silico analysis was also performed. The assay was shown to be specific, as no cross-reactions were observed either in vitro or in silico, and sensitive, with a 95% limit of detection ranging from 10-0,39 to 10-3,61 TCID50/reaction, for the detection of Simbu serogroup viruses. The repeatability of the assay was evaluated for both probes detection, using the intra- and inter-run standard deviations and coefficient of variation. This work resulted in a manuscript in submission process to a peer-reviewed journal. In addition, a comprehensive review of the viruses of the Simbu serogroup was carried out, including sites of viral isolation and seroconversion, and a map was generated using a geographic information system tool.<br>RESUMO - O serogrupo Simbu pertence ao género Orthobunyavirus, família Peribunyaviridae, e é constituído por 32 vírus de RNA tri-segmentado de cadeia simples e polaridade negativa, divididos em duas clades filogenéticas. Alguns membros deste grupo de vírus transmitidos por artrópodes, com distribuição cosmopolita, causam doença neurológica em humanos bem como doença reprodutiva e neurológica em animais domésticos, no entanto, o diagnóstico definitivo requer sempre confirmação laboratorial. Poucos ensaios de RT-PCR em tempo real têm sido desenvolvidos para o diagnóstico molecular destes vírus, ainda assim, existem dois que se destacam pela capacidade de detecção em largo espectro. Um deles, com sistema de detecção de fluorescência baseado na utilização de SYBR Green, é capaz de detectar vírus das duas clades, mas não é absolutamente específico, e o outro, baseando-se na utilização de sondas TaqMan, só detecta vírus de uma das clades. Um ensaio de RT-PCR em tempo real grupo-específico, inédito, com sondas TaqMan, foi desenvolvido, optimizado e caracterizado em termos laboratoriais, para a detecção em largo espectro dos orthobunyavirus do serogrupo Simbu. Os dados publicados referentes ao genoma dos membros do serogrupo foram analisados, e uma região conservada, situada no segmento codificante da proteína da nucleocápdise, foi eleita para o desenho de um par de primers específicos universal, bem como de duas sondas de hidrólise específicas, distintamente marcadas, e que, portanto, permitem a diferenciação entre as clades filogenéticas. Sete isolados de referência foram utilizados no desenvolvimento do ensaio, nomeadamente Akabane orthobunyavirus, Simbu orthobunyavirus, Shuni orthobunyavirus, Sathuperi orthobunyavirus, Shamonda orthobunyavirus, Ingwavuma virus e Sabo virus e, consequentemente, as concentrações dos primers e sondas na reação foram optimizadas. A eficiência de amplificação foi determinada para cada um dos vírus: AKAV (99%), SIMV (96%), SHUV (96%), SATV (97%), SHAV (84%), INGV (93%) e SABOV (110%). Um painel constituído por vírus geneticamente relacionados, agentes causais de aborto em ruminantes e transmitidos por artrópodes foi seleccionado no sentido de avaliar a especificidade do ensaio in vitro, tendo sido também efectuada uma análise in silico. O ensaio é específico, visto que não foram observadas reacções cruzadas quer in vitro quer in silico, e sensível, com um limite de detecção de 95% entre 10-0,39 a 10-3,61 TCID50/reacção, para a detecção de vírus do serogrupo Simbu. A repetibilidade foi avaliada para a detecção com ambas as sondas, pelo cálculo do desvio padrão intra- e inter-corridas bem como do coeficiente de variação. Este trabalho originou um manuscrito em processo de submissão para uma revista cientifíca. Além disso, foi levada a cabo uma revisão bibliográfica do serogrupo Simbu, incluindo locais de isolamento viral e seroconversão, e um mapa inédito desta distribuição foi gerado.<br>N/A
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Morar, Darshana. "The development of an interferon-gamma (IFNγ) assay for the diagnosis of tuberculosis in African elephants (Loxodonta africana) and black rhinoceros (Diceros bicornis)". Diss., University of Pretoria, 2003. http://hdl.handle.net/2263/23032.
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The objective of this project was to design tools for a diagnostic test that will prove valuable in the detection of tuberculosis in elephants and rhinoceros by using the cytokine IFNγ as an indicator of Mycobacterium bovis responsiveness. Interferon-gamma (IFNγ), a type II interferon, is a cytokine mainly produced by Th1 and cytotoxic T-cells expressing surface markers CD4 and CD8, respectively and natural killer cells (Ibelgaufts; 1999). In response to a mycobacterial infection antigen specific Th1- and cytotoxic T-cells are induced. When these cells encounter their specific mycobacterial antigen, they will respond by producing IFNγ. Based on this principle a diagnostic test was developed. In this test PBMCs will be stimulated with M.bovis specific antigen and the subsequent production of IFNγ by specific T-helper cells will be determined by IFNγ of elephants and rhinoceros. In order to develop such an assay recombinant elephant and rhinoceros IFNγ was cloned, sequenced, expressed, purified and subsequently a monoclonal antibody against IFNγ was produced. Monoclonal antibodies were selected by a number of ELISAs using recombinant IFNγ. Preliminary results are promising and further tests are underway regarding the specificity and sensitivity of the assay before field trials can be performed. The results of this study has significant implications in the design of an IFNγ diagnostic kit for the diagnosis of tuberculosis, as caused by M.bovis, in elephants and rhinoceros as well as other wildlife affected by this debilitating disease.<br>Dissertation (MSc (Veterinary Science))--University of Pretoria, 2003.<br>Veterinary Tropical Diseases<br>unrestricted
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Grushka, Daniel. "Development of a monoclonal-antibody based antigen detection enzyme linked immunosorbent assy (ELISA) for the diagnosis of human toxoplasmosis." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=32998.
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Although several commercial serological kits exist for Toxoplasma serodiagnosis, the unambiguous diagnosis of many clinically important Toxoplasma infections remains problematic. This is particularly true in establishing the timing of infection in pregnant women and in demonstrating reactivation of disease in immunocompromised hosts. The wide tissue tropism and distinct life-cycle stages of toxoplasmosis raise the possibility that the detection of circulating tachyzoite antigens may be of use in these situations. We have developed a series of antigen capture Enzyme Linked Immunosorbent Assays (ELISAs) using a panel of novel monoclonal antibodies (mAbs) directed against T. gondii tachyzoite antigens. Using a pool of these mAbs to capture whole tachyzoite lysate antigen in 'spiked' negative serum, the detection limit of our ELISA was 1--2mug/ml of protein. The sensitivity of this ELISA was 52% (n = 412). We postulated that our low sensitivity was mainly due to circulating immune complexes. This was confirmed by the disappearance of 'spiked' tachyzoite lysate antigen in antibody positive samples followed by the reappearance of antigen upon 12% trichloroacetic acid (TCA) treatment. Furthermore, the use of 12% TCA significantly increased antigen detection (p = 0.00001). Preliminary results suggested that assay sensitivity was 96% (n = 254) while assay specificity was 97% (n = 253). Early reports suggest that Toxoplasma antigen levels in serum are transient. The magnitude and kinetics of antigenemia with the specific Toxoplasma products recognized by our panel of mAbs remain to be determined. This optimized assay can now be used to test sera from otherwise healthy and immunocompromised subjects to determine its clinical utility.
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Sum, Siu-man Simon, and 岑紹文. "The development and assessment of assays for quantitation of hepatitisB virus DNA (HBV DNA) and the clinical significance of low HBV DNAlevel in patients with chronic hepatitis B." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B3013836X.
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Smith, Kathryn L. "DEVELOPMENT OF A NEW ALLELIC DISCRIMINATION REAL-TIME PCR ASSAY FOR THE DIAGNOSIS OF EQUINE HERPESVIRUS-1 AND CHARACTERIZATION OF THE VIRULENCE DETERMINANTS OF THE VIRUS." UKnowledge, 2013. http://uknowledge.uky.edu/gluck_etds/12.
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Equine herpesvirus-1 (EHV-1) can cause acute upper respiratory tract disease, abortion, neonatal death and neurological disease in horses. Rapid, accurate and timely diagnosis of EHV-1 infection in horses is important to curtail the spread of this pathogen. It has been reported that the neuropathogenic phenotype of EHV-1 can result from a single non-synonymous nucleotide substitution at position 2254 (A→G2254) in open reading frame 30 (ORF30). This was the basis for the development of an allelic discrimination, real-time PCR assay to distinguish between potential neuropathogenic and non-neuropathogenic EHV-1 strains. However, PCR analysis of a panel of EHV-1 abortion isolates revealed that other point mutations within ORF30 could produce false negative results with this previously described assay. Therefore, one of the objectives of this dissertation project was to develop a more sensitive and specific allelic discrimination real-time PCR assay for the detection of EHV-1. This was achieved by redesigning the primers and probes targeting ORF30. The new assay was ten times more sensitive than the original assay, with a lower detection limit of 10 infectious virus particles. While mutations within EHV-1’s genome can hinder diagnosis, they can also impact the virulence of the virus. Objective two, therefore, was to determine if sequential cell passage of T953 would induce sufficient attenuation of the EHV-1 genome to produce a low virulence phenotype. Two separate groups of 28 BALB/c mice were inoculated with either the parental strain or passage 135 (T953 P135) of EHV-1 strain T953. The animals were observed for fourteen days, euthanized and their tissues analyzed for the presence of EHV-1. At the conclusion of the fourteen day observation period, all of the mice infected with T953 P135 survived and regained their pre-inoculation body condition. Furthermore, there were significant differences in virus titer and viral DNA concentrations between T953 P135 and the parental strain, further confirming the attenuated phenotype of the virus. Taken together, data from this study clearly demonstrates that sequential cell culture passage of the neuropathogenic T953 strain of EHV-1 results in attenuation for young adult BALB/C mice.
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Bhattacharyya, T. "Parasite diversity and innovative serology : development of Trypanosoma cruzi lineage-specific diagnosis of Chagas disease and of prognostic assays for visceral leishmaniasis." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2015. http://researchonline.lshtm.ac.uk/2173663/.
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Trypanosoma cruzi and the Leishmania donovani complex are parasitic protozoa that, respectively, cause Chagas disease in the Americas, and visceral leishmaniasis, predominantly in South Asia, East Africa, and Brazil. T. cruzi is divided into the lineages TcI-TcVI. The relationship between infecting lineage(s) and spectrum of clinical presentations remains poorly understood. This project developed lineage-specific serology to identify an individual’s history of lineage infection. A high level of polymorphism in the surface mucin TSSA was identified, and lineage-specific synthetic peptides based on this diversity were applied here in ELISA with chagasic sera from endemic countries. Peptide TSSApep-II/V/VI, based on a sequence common to those lineages, was widely recognised by sera from Southern Cone countries, and also unexpectedly by four samples from Ecuador; TSSApep-V/VI, which differs by a single amino acid from TSSApep-II/V/VI, was also recognised in these regions. A single TSSApep-IV reaction was seen in both Colombia and Venezuela. However, TSSApep-I was rarely and weakly recognised among the serum panel. Among the Brazilian patients, a much higher proportion of TSSApep-II/V/VI responders had ECG abnormailities than non-responders (38% vs. 17%, p<0.0001). Rapid diagnostic tests for L. donovani complex infection based on rK39 antigen have lower sensitivity in East Africa compared to South Asia. The homologous sequences of rK39, and of another proposed diagnostic antigen HASPB, were amplified from a panel of East African L. donovani strains, and compared to published sequences, revealing significant diversity from rK39 and South Asian sequences, and non-canonical combinations of HASPB repeats. Cohorts of Indian and Sudanese VL patients were assayed by ELISA for anti- Leishmania IgG levels. There was an overall 46.8 – 61.7 fold lower response in the Sudanese cohort, as calculated by mean reciprocal log10t50 titres, regardless of antigen source, patient gender or age. An investigation into the association of IgG subclass reactivity with VL clinical status revealed significantly elevated IgG1 levels in patients with active (pre-treatment) VL and those with post-therapy relapse compared to those deemed to be cured. A novel prototype rapid immunochromatographic test to detect IgG1 gave > 80% of relapsed VL patients as IgG1 positive, and 80% of cured patients as IgG1 negative.
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Lu, Zhengchun. "DEVELOPMENT OF MOLECULAR DIAGNOSTIC ASSAYS FOR EQUINE RESPIRATORY VIRUSES AND ANALYSIS OF THE ROLE OF EQUINE ARTERITIS VIRUS ENVELOPE PROTEINS IN THE EARLY EVENTS OF VIRUS ENTRY." UKnowledge, 2012. http://uknowledge.uky.edu/gluck_etds/3.
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There is an urgent need for detection of viral respiratory pathogens to identify the causal agent(s) involved and to prevent the spread of related diseases. The first part of this dissertation focuses on development, optimization and validation of Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays for the detection of several common equine viral pathogens: equine arteritis virus (EAV), equine influenza virus and equine rhinitis viruses A and B. Emphasis of the second part of this dissertation is on studying the role of EAV envelope proteins in virus attachment and entry. Using an infectious cDNA clone of EAV and reverse genetics, a panel of chimeric viruses was generated by swapping the N-terminal ectodomains and full-lengths of the two major envelope proteins (GP5 and M) from porcine reproductive and respiratory syndrome virus (PRRSV). The recombinant viruses expressing the N-terminal ectodomain of PRRSV GP5 or M or both (GP5ecto, Mecto, and GP5&Mecto, respectively) in an EAV backbone were viable and genetically stable. Compared to the parental virus, these three chimeric viruses produced lower titers and smaller plaque sizes indicating that they have a crippled phenotype. Interestingly, the three chimeric viruses could only infect EAV susceptible cell lines but not the PRRSV susceptible cell line. Therefore, the exchange of GP5 and/or M protein N-terminal ectodomains from PRRSV did not alter the cellular tropism of the chimeric viruses. We also investigated the role of one of the minor envelope proteins (E) of EAV in virus attachment and entry. The results showed that EAV infection of equine endothelial cells is heparin-dependent and the Cterminus of the E protein contains a putative heparin-binding domain. We generated a panel of arginine to glycine mutations in the conserved region of both the full-length EAV infectious cDNA clone and individual E protein expression vectors. The triple mutation R52,60,65G construct grew significantly slower and produced much smaller plaques. The double mutant R52,60G completely blocked the interaction between E protein and heparin. Taken together, these data indicated that E protein interacts with heparin to facilitate virus attachment and plays a major role in EAV infection.
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Delport, Darnielle. "The development and application of a polymerase chain reaction (PCR) based assay to determine the impact of genetic variation in South African patients diagnosed with depression." Thesis, Stellenbosch : Stellenbosch University, 2014. http://hdl.handle.net/10019.1/86564.
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Thesis (MPath)--Stellenbosch University, 2014.<br>ENGLISH ABSTRACT: Major Depressive Disorder (MDD) is a severe debilitating medical condition that may lead to suicide. Due to a poor understanding of the biological mechanisms underlying the disease process therapeutic decisions are usually taken using a ‘trial and error’ approach. This is not ideal since many treatments do not work as expected for all individuals. Studies have shown that only half of MDD patients receive the appropriate treatment, whereas many patients have adverse response to anti-depressants. These may include weight gain and raised homocysteine levels that may further compromise the health status of MDD patients and may partly explain the link with cardiovascular disease.
The objective of the study was to identify genetic risk factors interacting with environmental factors implicated in MDD that may be of relevance to the South African population. Polymorphisms in the MTHFR (677 C>T, rs1801133 and 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) and SLC6A4 (43 bp ins/del, rs4795541) genes were genotyped in 86 MDD patients and 97 population-matched controls. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the selected SNPs against direct sequencing as the gold standard for 2) possible integration into a pathology-supported genetic testing strategy aimed at improved clinical management of MDD. A total of 183 unrelated Caucasians participated in the study, including 69 females and 17 males with MDD and 57 female and 40 male controls without a personal and family medical history of overlapping stress/anxiety and depressive disorders. All study participants were genotyped for the six selected SNPs considered clinically useful based on international data. The allelic distribution of the SNPs, single or combined into a genotype risk score after counting their minor alleles, did not differ between MDD patients and controls. Homocysteine levels were determined and correlated with body mass index (BMI) and other variables known to influence these phenotypes. The folate score assessed with use of the study questionnaire was significantly lower in the patient group compared with controls (p=0.003) and correlated significantly with BMI, particularly in females (p=0.009). BMI was on average 8% higher in the MDD patients compared with controls (p=0.015) after adjustment for age and sex. The MTHFR rs1801133 677 T-allele was associated with a 14% increase in BMI in MDD patients but not controls (p=0.032), which in turn was associated with significantly increased homocysteine levels (p<0.05).
The aims of the study were successfully achieved. Identification of the MTHFR rs1801133 677 T-allele reinforces the importance of adequate folate intake in the diet due to increased risk of obesity and depression found to be associated with low dietary intake. Evidence of shared genetic vulnerability for many chronic diseases and drug response mediated by the MTHFR 677 T-allele support the clinical relevance of this low-penetrance mutation.<br>AFRIKAANSE OPSOMMING: Major depressie (MD) is ‘n aftakelende siektetoestand wat tot selfdood kan lei. Onkunde oor die siekte se onderliggende biologiese meganismes lei dikwels tot ‘n lukrake terapeutiese benadering. Dit is ‘n onbevredigende situasie aangesien indiwidue verskillend reageer op die middels wat voorgeskryf word. Navorsing toon dat slegs ongeveer die helfte van MD pasiënte toepaslike behandeling kry, terwyl anti-depressante ‘n nadelige uitwerking het op baie pasiënte. Dit sluit massatoename en verhoogde homosisteïenvlakke in wat MD pasiënte se gesondheid bykomend nadelig kan beïnvloed en die verband met kardiovaskulêre siekte gedeeltelik kan verklaar.
Hierdie studie poog om MD verwante genetiese risikofaktore en omgewingsfaktore wat mekaar beïnvloed en moontlik op die Suid Afrikaanse bevolking betrekking het, te identifiseer. Polimorfismes in die MTHFR (677 C>T, rs1801133 en 1298 A>C, rs1801131), COMT (472G>A, rs4680), CYP2D6 (6937G>A, rs3892097), ASMT (24436 G>A, rs4446909) en SLC6A4 (43 bp ins/del, rs4795541) gene is geanaliseer in 86 MD pasiënte en 97 kontroles geselekteer van dieselfde populasie. Die spesifieke doelwitte was om 1) hoë deurset direkte polimerase kettingreaksie (RT-PCR) genotiperingstoetse vir die 6 gekose polimorfismes met direkte volgordebepaling as maatstaf analities te valideer vir 2) moontlike insluiting in ‘n patologie-ondersteunde genetiese toetsstrategie met die oog op beter kliniese hantering van MD. Altesaam 183 Kaukasiërs het aan die studie deelgeneem. Die MD pasiënte het uit 69 vroue en 17 mans bestaan. Die kontroles (57 vroue en 40 mans) het geen mediese geskiedenis (persoonlik of familie) van oorvleuelende stress/angstigheid of depressie gehad nie. Gebaseer op internasionale data, is al die deelnemers vir die 6 gekose, potensieel klinies-bruikbare polimorfismes getoets. Die alleliese verspreiding van die polimorfismes enkel of gekombineer (uitgedruk as ‘n genotipe-risiko-syfer nadat minor allele getel is), was dieselfde in MD-pasiënte en kontroles. Homosisteïenvlakke is bepaal en gekorreleer met die liggaamsmassa-indeks (BMI) en ander veranderlikes wat bekend is vir hulle invloed op hierdie fenotipes. In teenstelling met die kontroles, was die folaat telling, soos bepaal met die studievraelys, betekenisvol laer in die pasiënte (p=0.003). Die korrelasie met die liggaamsmassa-indeks, spesifiek by vroue, was ook betekenisvol (p=0.009). Na aanpassings vir ouderdom en geslag, is gevind dat die liggaamsmassa-indeks gemiddeld 8% hoër was in die die MD pasiënte teenoor die kontroles. By MD-pasiënte, maar nie by die kontroles nie, is die MTHFR rs1801133 677 T-alleel geassosieer met ‘n 14% toename in liggaamsmassa-indeks (p=0.032), wat ook geassosieer was met betekenisvolle verhoogde homosisteïenvlakke (p<0.05).
Die doelwitte van die studie is bereik. Identifisering van die MTHFR rs1801133 677 T-alleel beklemtoon hoe belangrik dit is om voldoende folaat in te neem, veral omdat ‘n verhoogde risiko vir vetsug en depressie met ‘n lae folaatinname in die diet geassosieer word. Die kliniese belang van die MTHFR 677 T-alleel word beklemtoon deur toenemende bewyse wat daarop dui dat gedeelde genetiese vatbaarheid vir ‘n verskeidenheid van kroniese siektes asook middelrespons aan bemiddeling deur hierdie lae penetrasie mutasie toegeskryf kan word.<br>Winetech<br>Technology for Human Resources and Industry Program (THRIP).
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Arap, Marco Antonio. "Estudo da proteína de choque térmico GRP78 para o desenvolvimento de um sistema de receptor-ligante para o câncer de próstata." Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/5/5153/tde-31052007-122749/.
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Introdução: Apesar dos avanços nas técnicas de diagnóstico e tratamento, o câncer de próstata avançado ainda é uma condição letal. Terapêuticas mais eficazes são necessárias para reduzir as taxas de morbi-mortalidade associadas à doença. A Proteína-78 regulada pela glicose (GRP78), uma proteína de choque térmico envolvida na apresentação de antígenos, foi recentemente descrita como sendo um possível marcador molecular para o câncer de próstata. Ainda mais, a resposta imune a essa proteína mostrou correlação com o desenvolvimento de doença hormônio-independente e com pior sobrevida para a doença. Objetivos: Neste estudo, avaliou-se a hipótese de que a GRP78 poderia ser usada como marcador molecular em câncer de próstata no desenvolvimento de um sistema de receptor-ligante, através do uso da tecnologia de apresentação de fagos. Casuística e métodos: Inicialmente, foram clonados dois peptídeos que apresentam afinidade à proteína regulada pela GRP78 (os peptídeos WIFPWIQL e WDLAWMFRLPVG) no vetor fUSE5, criando-se fagos com capacidade teórica de ligação à mesma proteína. Posteriormente foi testada a capacidade de ligação desses fagos à GRP78 na membrana de células prostáticas malignas em solução, em xeno-tumores in vivo e em metástases ósseas de câncer de próstata humano. Resultados: Demonstrou-se que ambos os fagos se ligam especificamente à GRP78 in vitro, em comparação à proteínas com seqüência semelhante (proteínas de choque térmico 70 e 90) e não semelhante (albumina sérica bovina). Em seguida, mostrou-se que esses fagos se ligam com afinidade pelo menos 30 vezes maior à células de câncer de próstata que o fago controle, e que os fagos são internalizados por essas células. Posteriormente, mostrou-se que os fagos rastrearam xeno-tumores prostáticos quando injetados in vivo num modelo animal de câncer de próstata. Finalmente, mostrou-se que os fagos ligam-se especificamente à GRP78 expressa em metástases ósseas de adenocarcinoma prostático humano. Conclusões: Os fagos criados apresentam capacidade de ligação específica à GRP78 in vitro, em células em suspensão e in vivo. A estratégia e o sistema de receptor-ligante definidos no presente estudo podem ter implicacões relevantes no desenvolvimento de terapias dirigidas para o tratamento do câncer de próstata.<br>Introduction: Despite the advances in diagnosis and treatment, advanced prostate cancer remains a lethal condition. Improved methods of therapy are needed to reduce the morbidity and mortality rates associated with this disease. The Glucose-regulated protein-78 (GRP78), a stress-responsive heat-shock protein involved in antigen presentation, was recently described as a possible molecular marker for prostate cancer. Moreover, immune response against this protein was shown to have correlation with the development of androgen-independent prostate cancer and shorter overall survival. Objectives: We hipothesized that GRP78 could be used as a molecular marker for prostate cancer in the development of a receptor-ligand system, by using phage display technology. Patients and methods: We initially cloned two GRP78-targeting peptides (WIFPWIQL and WDLAWMFRLPVG) into a fUSE5-based phage. We then tested binding capacity of the phage to GRP78 in vitro, to GRP78 expressed in intact prostate cancer cell membranes, to a prostate cancer xenograft and to human bone metastases. Results: We showed that both phage created bound specifically to GRP78 in vitro, in comparison to related (Heat-shock proteins 70 and 90) and unrelated control proteins (bovine serum albumin). Next, we showed that these phage bound at least 30 times more to prostate cancer cells than the control phage, and were also internalized into these cells. Both GRP78-binding phage showed a strong homing in vivo to a human prostate cancer xenograft in a mouse model. Finally, we showed that both phage bound specifically to GRP78 expressed in human prostate cancer bone metastases. Conclusions: Both phage are capable of binding specifically to GRP78 in vitro, in the context of intact prostate cancer cells and in vivo. The strategy and the ligand-receptor system we have defined in this study may have relevant implications in the development of targeted therapies for the treatment of prostate cancer.
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Yiing, Shyh-Shuan, and 殷世栓. "Development of enzyme-linked immunosorbent assay diagnostic kit for fowl cholera in Taiwan." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/14044381933318672448.
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碩士<br>國立屏東科技大學<br>獸醫學系<br>93<br>The purpose of this research is to develop an ELISA kit applied to dia- gnose the fowl cholera. CHC-00*01 antigen which is the combination of CHC-00 and CHC-01 strain through Indirect ELISA Test, is compared with the antigen mixed with KSC-98 or KSD-99 strain, in order to detect the ab- sorbent rate of the fowl serum. The result of the biochemical evaluation uti- lizing the Biolog''s GN2 MicroPlateTM points out that 99% are Pasteurella multocida bacterium. The cross infective test shows the interactive infection of P. multocida strain between the chicken and the duck. Box titration is uti- lized to gain the ratio of the antigen and the antibody, and is applied 96-wells ELISA plate as the positive and negative controlling group. The final result indicates that, based on 625 nm wave length, P. multocida binary antigen the absorbent rate is 0.25, is diluted into 1：512 and it is the best ratio that the serum from the chicken is diluted into 1：64. According to the ratio, the OD ( optical density ) value of the positive controlling group of the chicken seru- m is 0.416; the negative one is 0.100. The ratio of the positive and negative of the chicken serum is 0.416/0.100＝4.16≥ 4, which is the distinctive varie- ty. Duncan''s multiple range test for variable is applied for analyzing the resu- lt and it shows that there are the distinctive variety ( P＜0.01 ) between CHC -00*01 antigen and CHC-00*01*KSC-98 antigen; the distinctive variety bet- ween ( P＜0.01 ) CHC-00*01 antigen and CHC-00*01*KSD-99 antigen.The result of applying the ELISA kit to detect the serum of 200 chickens from the fields provides that the positive rate of the chicken serum is 29.5% ( 59/200 birds ) and it is coordinated with the clinical symptom. After 141 known gro- ups with the negative serum sample are tested, if the range of S/N ratio is on 1.0; the reliability of the positive and negative serum is 99% confidence level. i. e., the single absorbent rate is diluted into 1：64; S/N ratios is limited on ＞1.0; defined as a positive serologic response; with the acknowledgment of a 1% false positive error rate at S/N＝1.0. This homologous antigen which is the combination of CHC-00 and CHC-01 strain could be the best antigen for preparing ELISA Kit in improving the diagnosis of fowl cholera immunorea- ctions or infections of chickens in Taiwan.
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Chou, Chih-Lin, and 周知林. "Development of a Molecular Assay for Laboratory Rodent Fur Mite Diagnosis and Comparison with the Traditional Diagnostic Methods." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/8jgkve.
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碩士<br>國立臺灣大學<br>分子暨比較病理生物學研究所<br>107<br>Rodent fur mite infestation is a persistent and intractable problem in laboratory rodent colonies, due to insensitive diagnostics, unrepresentative samples for testing, and improper sentinel system. To improve the sensitivity and efficiency of fur mite detection, a multiplex PCR assay was developed to simultaneously detect and differentiate different species of fur mites, including Myocoptes musculinus (COP), Myobia musculi (MOB) and/or Radfordia spp. (RAD), and species A (SPA; a novel rodent fur mite identified in Taiwan), with the existence of a rodent housekeeping gene. This multiplex PCR could specifically detect as low as 10 copies of each species in equal-amount triple infestation. Super-infestation with 10 to 100-fold differences in mite burdens could be also detected. In comparison of the multiple PCR and traditional methods (pluck test, tape test, and pelt exam) for fur mite diagnosis, 48 rodents and 25 cage environment samples were evaluated for the fur mite infestation. In screening the status of various fur mites on individual animals, the multiplex PCR assay showed distinctly higher in sensitivity and accuracy (86 % and 95.1 %) than that of traditional methods (sensitivity: 6 % - 46 %, accuracy: 67.4 % - 81.3 %). Interestingly, by using cage wipe environmental samples, the multiplex PCR assay exhibited 100 % in both sensitivity and accuracy on the fur mite detection and differentiation. The COP/MOB-RAD/SPA/Actin multiplex PCR assay developed in this study could be a reliable alternative method for routine pathogen monitoring (animal or environment) or for tracing the suspect fur mite outbreak in rodent colonies.
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Mashinini, Bongiwe. "Development of a diagnostic ELISA for the hepatitis B x-protein using monoclonal antibodies." Thesis, 2010. http://hdl.handle.net/10539/8803.
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MSc (Med), Faculty of Health Sciences, University of the Witwatersrand<br>The hepatitis B virus remains a major public health problem even after decades of its discovery. Horizontal transmission during early childhood is the predominant mode of transmission in highly endemic regions such as sub-Saharan Africa. Infection exhibits a wide spectrum of clinical manifestations, from an asymptomatic stage to severe liver disease which may result in hepatocellular carcinoma (HCC). The HBV X protein (HBx) has been implicated in carcinogenesis, which often has a poor prognosis, consequently the use of highly specific monoclonal antibodies (mAbs) directed against HBx in an enzyme-linked immunosorbent assay (ELISA) could lead to early identification of HBV carriers at risk of developing liver cancer. A variety of mixed hybridoma cell cultures secreting anti-HBx antibodies were cloned and sub-cloned by “limiting dilution”. Clonal supernatants were assessed for anti-HBx antibody production by Indirect ELISA and Western/Immunoblotting. Monoclonal antibodies were then characterized according to their relative binding affinity (Indirect ELISA) and relative epitope specificity (Competitive ELISA). One of our monoclonal antibodies was found to bind to the same epitope on HBx as the commercial anti-HBx antibody and with the same high affinity.
In the developed Sandwich ELISA, our monoclonal antibody proved effective as the „detecting‟ antibody when the commercial anti-HBx antibody was deployed as the
„capture‟ antibody. This Sandwich ELISA will be further developed in our laboratory with the object of applying it to patient sera.