Luminescent semiconductor quantum dots (QDs) are extensively researched for use in biological applications. They have unique optical and physical properties that make them excellent candidates to replace conventional organic dyes for cellular labeling, multiplexing, nucleic acid detection, and as generalized probes. The primary focus of this dissertation was to utilize quantum dots for improvement in immunoassays. Specifically, atherosclerosis biomarkers were detected simultaneously in an effort to demonstrate advances in early detection diagnostics. Quantum dot-antibody bioconjugates were prepared by encapsulation into mesoporous silica and functionalized with thiol and amine groups to enable bioconjugation. Functionalization of the mesoporous silica quantum dot composites facilitated biocompatibility for use with biological buffers in immunoassays. These bioconjugates were used in a sandwich immunoassay to detect atherosclerosis biomarkers IL-15 and MCP-1. Sandwich assays employ capture antibodies immobilized onto a well plate to bind as much of the antigen as possible. The capture antibodies increased binding by at least 4 times the amount of antigen bound to the surface of a direct detection assay. The sandwich immunoassay was able to detect 1 pg/mL of IL-15 and 50 pg/mL of MCP-1 biomarkers. Human serum albumin nanoparticles (HSAPs) were synthesized via a desolvation and crosslinking method. Human serum albumin is a versatile protein being used in a variety of applications. Quantum dots were loaded into HSAPs as potential detection probes for immunoassays. Efficient loading was not achieved, and the assay was unable to improve current detection limits. Controlled release studies were explored using HSAPs loaded with superparamagnetic iron oxide nanoparticles and a fluorescent drug analog. Exposure to a magnetic field resulted in degradation of the HSAPs. The fluorophore was released and measured to examine how cancer drugs might be controlled through a magnetic field. Gold nanorods and an anticancer drug, Sorafenib, were also encapsulated into HSAPs for treatment of renal cell carcinoma in vivo. Laser irradiation treatment combined with Sorafenib resulted in 100% tumor necrosis and total elimination of any viable tumor present. HSAPs have demonstrated remarkable potential as drug delivery nanocarriers.

Introduction: Pulmonary Surfactant reduces surface tension in the terminal airways thus facilitating breathing and contributes to host's innate immunity. Surfactant Proteins (SP) A, B, C and D were recently identified as inherent proteins of the CNS. Aim of the study was to investigate cerebrospinal fluid (CSF) SP levels in hydrocephalus patients compared to normal subjects. Patients and methods: CSF SP A-D levels were quantified using commercially available ELISA kits in 126 patients (0±84 years, mean 39 years). 60 patients without CNS pathologies served as a control group. Hydrocephalus patients were separated in aqueductal stenosis (AQS, n = 24), acute hydrocephalus without aqueductal stenosis (acute HC w/o AQS, n = 16) and idiopathic normal pressure hydrocephalus (NPH, n = 20). Furthermore, six patients with pseudotumor cerebri were investigated. Results: SP AÐD are present under physiological conditions in human CSF. SP-A is elevated in diseases accompanied by ventricular enlargement (AQS, acute HC w/o AQS) in a significant manner (0.67, 1.21 vs 0.38 ng/ml in control, p<0.001). SP-C is also elevated in hydrocephalic conditions (AQS, acute HC w/o AQS; 0.87, 1.71 vs. 0.48 ng/ml in controls, p<0.001) and in Pseudotumor cerebri (1.26 vs. 0.48 ng/ml in controls, p<0.01). SP-B and SP-D did not show significant alterations. Conclusion: The present study confirms the presence of SPs in human CSF. There are significant changes of SP-A and SP-C levels in diseases affecting brain water circulation and elevation of intracranial pressure. Cause of the alterations, underlying regulatory mechanisms, as well as diagnostic and therapeutic consequences of cerebral SP's requires further thorough investigations.

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.

Thesis (Ph.D.(Public Health))--University of the Witwatersrand, Faculty of Health Sciences, 2012. / Measuring HIV incidence has proved challenging over the years. A number of serological HIV assays have been proposed, and among these, the BED Capture Enzyme Immunoassay (CEIA) is one of the more widely used. Although the assay performs well among known seroconverting panels, it has been shown to classify some long term infected patients as being recently infected. Information on the performance of the BED assay among low CD4 cell count patients and those on antiretroviral therapy is limited. The risk of onwards transmission of HIV has been reported to be elevated around the seroconversion period compared to the chronic stage of infection. RNA viral load has been reported as the strongest predictor of HIV transmission compared to other HIV markers. Understanding how these markers influence the relationship between the likelihood of being recently infected and the BED assay might help in understanding some of the shortcomings of the BED assay. The main aim of this study was to understand the properties of the BED assay. The performance of the BED assay among advanced HIV disease patients and the influence of ART on BED levels once patients started treatment was investigated. The BED assay and CD4 cell count were used to quantify the risk of in utero and intrapartum transmission to their infants among women believed to have seroconverted during pregnancy. The influence of viral load, haemoglobin and mid-upper arm circumference was investigated on the relationship between the probability of being recently infected and BED ODn levels. Methods Cryopreserved plasma samples from HIV patients on the national antiretroviral treatment (ART) rollout programme at Tygerberg Hospital HIV clinic, South Africa, iv were used to investigate the effect of ART on BED ODn levels once patients commenced treatment. Mixed effect logistic regression models accounting for multiple readings per patient were used. To investigate the risk associated with seroconversion during pregnancy HIV seropositive women who had just given birth were classified into mutually exclusive groups according to their likelihood of having recently seroconverted using BED and CD4 cell count levels. Multinomial logistic regression models adjusting for other factors were used to assess the risk of MTCT in utero and intra-partum infection comparing these groups. To investigate the relationship between BED ODn levels and the probability of being recently infected, BED data from known HIV infected women and women who seroconverted over a 2 year period was used. Fractional polynomial regression models that allow for non-linear functions to be fitted were used, and the influence of viral load, haemoglobin and mid-upper arm circumference was assessed through multi-variable models. Data from the Zimbabwe Vitamin A for Mothers and Babies (ZVITAMBO) project, a double blinded treatment-placebo trial was used for these last two objectives. Results Patients with very low CD4 cell counts were more likely to test false recently infected according to the BED assay than other patients. ART changed BED ODn kinetics among HIV patients on treatment. Over half of advanced disease stage patients were likely to be classified as being recently infected according to the BED assay 2 years into ART treatment. v Women who seemed to have seroconverted during pregnancy had elevated risk of transmitting HIV in-utero compared to chronic HIV patients. BED and CD4 cell count were not predictive of risk of intra-partum infections attributed to seroconversion during pregnancy. The relationship between the probability of being recently infected with HIV and BED ODn levels was described better using Fractional Polynomial regression models than using a linear model in BED ODn or a model in which the BED ODn was categorised. Viral load and haemoglobin were important independent predictors of incident infections. Conclusions If the BED assay is to be used for HIV incidence estimations patients on ART should be accounted for. The BED assay together with other HIV serological markers can be used as prognostic tools to assess the risk of HIV transmission. The risk of in-utero transmission of HIV is higher among women who seroconvert during pregnancy. Repeat HIV testing among pregnant women may help in identifying women who seroconvert during pregnancy, and these women will benefit from Prevention of Mother-to-Child transmission (PMTCT) programmes. It was found that additional markers such as viral load and haemoglobin did not alter the relationship between the probability of having been recently infected and BED ODn.

A toxoplasmose, protozoose disseminada de baixa morbidade, apresenta número significativo de doença ocular, congênita ou do sistema nervoso central. O diagnóstico é sorológico por diferentes testes, mas limiares baixos e variação individual levam a frequentes problemas. Novos imunoensaios fluorescentes de fase sólida (FLISA) usam a quantificação direta de anticorpos. Aqui, desenvolvemos um FLISA multiplex (FLISAm) para a detecção simultânea de anticorpos IgG e IgM contra Toxoplasma gondii. Após padronização, a eficiência do FLISAm com conjugados comerciais foi feita inicialmente de forma isolada para cada imunoglobulina em 140 amostras de soro de universitários previamente analisadas pelo ELISA IgG/IgM. FLISA IgG mostrou boa concordância (Kappa=0,7088), com sensibilidade de 83,3% e especificidade de 94,2%, enquanto FLISA IgM apresentou boa concordância (K=0,6026), menor sensibilidade de 55,5% e igual especificidade de 98,4%. Foram produzidos novos conjugados fluorescentes de maior especificidade e seu desempenho no FLISAm foi validado em 24 amostras e sua eficiência foi avaliada em 120 amostras conhecidas de soro de gestantes. FLISAm mostrou excelente concordância, tanto para a detecção de anticorpos IgG (K=0.8837, sensibilidade=100,0%, especificidade=87,5%), quanto para a detecção de anticorpos IgM (K=0,9187, sensibilidade=100%, especificidade=99,1%) com excelente reprodutibilidade. O teste desenvolvido é rápido, econômico, de fácil execução, alto rendimento e que pode ser utilizado como método de triagem de soroconversão em mulheres grávidas, útil em aplicações de grande número de amostras como o cuidado pré-natal. / Toxoplasmosis, a disseminated low morbidity protozoan disease, presented significant numbers of affected people, mainly ocular disease, fetal infections or encephalitis in immune deficient patients. Serology is the main diagnosis with commercial antibody assays, but individual variation or low thresholds cause many inconsistencies. New solid phase immunofluorescence assays (FLISA) allows direct antibody quantification in microplates. Here, we developed a multiplex FLISA (FLISAm) for simultaneous detection of IgG and IgM anti-Toxoplasma gondii antibodies. After standardization, the efficiency of this method was initially analyzed in isolated FLISA for each immunoglobulin with commercial conjugates in 140 serum samples of the students previously screened by IgG/IgM ELISA. IgG FLISA showed good concordance (Kappa=0.7088), 83,3% sensitivity and 94,2% specificity, while IgM FLISA also showed good concordance (Kappa=0,6026), lower 55,5% sensitivity and similar 98,4% specificity. New higher efficiency conjugated were prepared and tested in 120 serum samples of the pregnant woman in a same well conjunct IgG/IgM FLISAm. We also validate the FLISAm in 24 serum samples. Compared to isolated ELISA IgG/IgM, FLISAm demonstrated excellent concordance for IgG (Kappa=0.8837; sensitivity=100%; specificity=87,5%,) and IgM (Kappa=0,9187; sensitivity=100%; specificity=99,1%), with excellent reproducibility. The standardized FLISAm is quick, inexpensive, easily performed and high throughput and the assay can be used for screening serum conversion in pregnant women, useful in large numbers applications as antenatal care.

Neste trabalho, anticorpos anti-IgGh foram conjugados às nanopartículas de prata (NPAg) para detectar imunoglobulina G humana (IgGh). Um imunoensaio colorimétrico baseado na diminuição da agregação devido ao aumento da repulsão eletrostática após a interação ligante-alvo. A agregação é induzida pela variação da força iônica e uma mudança da coloração da suspensão coloidal de amarelo para vermelho pode ser observada. Na presença de IgGh, a agregação é inibida e a coloração da suspensão coloidal não se altera. As nanopartículas foram obtidas por meio de cinco procedimentos diferentes e caracterizadas por espectroscopia UV-Vis, espalhamento dinâmico de luz, difração de raios-X e microscopia eletrônica. Glicose e borohidreto de sódio foram utilizados como agentes redutores, enquanto CTAB e &#946;-ciclodextrina foram utilizados como estabilizantes. Citrato de sódio foi utilizado como agente redutor e/ou estabilizante. Nanoesferas de carbono foram obtidas por tratamento hidrotérmico de uma solução aquosa de glicose e também foram utilizadas no preparo das nanopartículas. As nanopartículas foram funcionalizadas com ácido mercaptossuccínico e a conjugação ocorreu devido à interação entre grupos aminas e grupos carboxílicos ionizados, presentes no anticorpo e agente de acoplamento, respectivamente. A estabilidade dos conjugados e o efeito da adição de IgGh foram avaliados para todos os sistemas preparados. As nanopartículas de prata preparadas com borohidreto de sódio e citrato de sódio foram selecionadas para serem aplicadas no desenvolvimento do imunoensaio e as condições experimentais foram avaliadas. Em condições ótimas, observou-se uma correlação linear entre a diminuição da agregação do sistema (NPAg-anti-IgGh) e a concentração de IgGh (0 a 200 ng mL-1). O limite de detecção foi estimado em 25 ng mL-1. O método colorimétrico apresentou boa seletividade para a detecção de IgGh. Além disso, foi obtido um resultado satisfatório ao aplicar o método para determinação do fator IX de coagulação. Foi desenvolvido também um método para determinação de ATP baseado na agregação de nanopartículas de ouro. Aptâmeros foram utilizados como elemento de reconhecimento. Em princípio, o método pode ser aplicável à determinação de outros analitos, por meio da substituição do aptâmero utilizado neste trabalho pelo oligonucleotídeo específico para o alvo de interesse. / In this work, antibodies to human immunoglobulin G (anti-IgGh) were used in combination with silver nanoparticle (NPAg) to detect IgGh. A colorimetric immunoassay based on the decrease of aggregation due to increased electrostatic repulsion upon ligand-target interaction. Aggregation was induced by varying the ionic strength and the solution of NPAg-anti-IgGh shows obvious visible color change from yellow to red. In the presence of IgGh aggregation the nanoparticle is inhibited and coloration of the colloidal solution does not change. The nanoparticles were obtained using five different procedures and they were characterized by UV-Vis spectroscopy, dynamic light scattering, X-ray diffraction and electron microscopy. Glucose and sodium borohydride were used as reducing agent, as CTAB and &#946;-cyclodextrin reagents were used as stabilizers. Sodium citrate was used as reducing and stabilizing agent. Carbon nanospheres were prepared by hydrothermal treatment of glucose and used in the preparation of NPAg. The nanoparticles were functionalized with dimercaptosuccinic acid and their conjugation occurred due to the interaction of positively charged amine groups and anionic groups (-COO-) present on the antibody and coupling agent. The stability of conjugates and the variation of aggregation in the presence of IgGh were evaluated for all systems. The NPAg prepared by sodium borohydride were selected for use in the immunoassay and the optimum conditions of the assay were investigated. Under the optimal conditions, the ration between the absorbance at 396 nm and 564 nm was linearly proportional to the IgGh concentration on a range from 0 to 200 ng mL-1, with a detection limit of 25 ng mL-1. The colorimetric method showed good selectivity for IgGh detection. It was possible to adapt the method to the determination of other proteins, such as factor IX. In another approach, anti-aptamer ATP was used to develop a colorimetric method for the determination of ATP based on stabilization of gold nanoparticles provided by strands of DNA.The strategy was based on stabilization of nanoparticles due to interaction with single strands of DNA, and the change of the stability of the nanoparticles provided by the conformational change of the aptamer following recognition. This method could in principle be used to detect analytes by substituting the aptamer used in this study by the specific aptamer for the target of interest

O objetivo deste trabalho foi avaliar de maneira detalhada e sistemática os perfis hormonais sanguíneos, fecais, urinários e salivares das progestinas e estrógenos durante o ciclo estral induzido de ovinos. Foram colhidas amostras diárias durante um período de 60 dias de sete fêmeas adultas (n=8) saudáveis e sexualmente maduras. Antes do início da fase de colheita das amostras, todos os animais foram submetidos ao protocolo de tratamento hormonal para indução e sincronização do cio durante dozes dias. O primeiro ciclo ovariano de cada animal desse experimento, detectado logo após a indução do cio foi descartado e seus valores não foram utilizados nas análises hormonais, pois poderiam estar sob o efeito dos hormônios exógenos. A concentração dos progestágenos foi determinada pelas técnicas analíticas de Radioimunoensaio (RIE) e Enzimaimunoensaio (EIE) e os estrógenos por RIE. Houve correlações entre as concentrações de progesterona medidas nas matrizes sérica e fecal, sérica e salivar, fecal e salivar (r=0,90, p<0,0001; r=0,90, p<0,0001; r=0,92, p<0,0001, respectivamente) durante os ciclos estrais observados (n=15). Obtivemos correlação (r=0,74, p<0,0001) entre as concentrações dos estrógenos quantificados nas matrizes sérica e fecal, mas não entre estas concentrações e aquelas medidas na matriz salivar. Não obtivemos nenhuma correlação entre as concentrações medidas na matriz urinária com as quantificadas nas outras matrizes para nenhum dos hormônios estudados. Obtivemos correlação entre as concentrações de progesterona medidas na matriz fecal pelos métodos de RIE e EIE (r=0,78, p<0,0001) e também na matriz salivar pelos dois métodos empregados (r=0,81, p<0,0001). Os resultados do presente experimento indicam que os imunoensaios utilizados podem ser utilizados para a avaliação das concentrações de progestágenos nas matrizes fecal e salivar durante o ciclo estral em ovinos. / The aim of the present work was evaluate the hormonal profiles of progestins and estrogens in blood, feces, urine and saliva during the induced estral cycle in ovine. Samples were collected daily a 60-day period from eight adult (n=8) cycling ewes. The animals were previously submitted to a protocol of estrus induction and synchronization for twelve days. In order to avoid the effect of exogenous hormones, the first cycle immediately after the synchronization was not considered for hormonal analysis. Progestagen concentrations were quantified by two analytical techniques, radioimmunoassay (RIA) and enzyme immunoassay (EIA). Estrogen concentrations were assessed by radioimmunoassay. Correlations in progesterone concentrations were found to be significant for serum and feces, serum and saliva and feces and saliva (r=0.90, p<0.0001; r=0.90, p<0.0001; r=0.92, p<0.0001, respectively) during the estrous cycles (n=15). Estrogen concentrations in the serum and feces were also positively correlated (r=0.74, p<0.0001). Salivary concentrations of estrogens were not correlated with fecal or serum concentrations of the same hormone. No correlation was found between urinary concentrations and concentrations found in other matrixes for both progestagens and estrogens. Concentrations of progestagens obtained using RIA and EIA were correlated on feces (r=0.78, p<0.0001) and saliva (r=0.81, p<0.0001). Results indicate that both immunoassays used in the present experiment can be used to evaluate progestagen concentrations on fecal and salivary matrixes during the estrous cycle of sheep.

A leptospirose é uma doença bacteriana infectocontagiosa, de curso agudo ou crônico, causada por espiroquetas do gênero Leptospira, de caráter zoonótico e cosmopolita que acomete o homem e os animais domésticos e silvestres. Pode ser transmitida de forma direta pelo contato com os fluidos contendo leptospiras, através das vias transplacentária e hematogênica, genital e o ato de mamar; ou de forma indireta pelo contato com ambiente contaminado com leptospiras. O conhecimento da gravidade da infecção, da distribuição geográfica, dos fatores de risco e das estirpes circulantes é de extrema importância para o estabelecimento da epidemiologia e o aprimoramento de medidas preventivas e diagnósticas. Neste estudo, avaliou-se a utilização de proteínas recombinantes de Leptospira spp. como antígenos no desenvolvimento de um teste rápido baseado em ensaio imunocromatográfico do tipo dipstick, como método diagnóstico da leptospirose animal. Foram selecionadas 11 proteínas recombinantes como candidatos a antígenos. As proteínas recombinantes purificadas foram avaliadas na detecção de anticorpos específicos por Western-blotting e ELISA. Somente a LipL32 apresentou reatividade com os soros positivos para leptospirose. Dois testes imunocromatográficos, utilizando a proteína LipL32, foram desenvolvidos. Um teste tipo I utilizando a proteína LipL32 conjugada ao ouro coloidal e outro tipo III com o ouro coloidal conjugado a proteína A e a LipL32 na linha teste. Em ambos as linhas teste e controle reagiram, demonstrando que os testes estão funcionando. O teste tipo I realizado manualmente mostrou resultados satisfatórios para os soros bovinos e soro hiperimune de coelho. O tipo III mostrou reatividade para as amostras de soro bovino, equino, coelho e cão. Quando se aplicou este dois testes em máquinas automatizadas não foi possível detectar a linha teste, provavelmente devido a necessidade de nova padronização de todos os parâmetros do método. / Leptospirosis is an infectious bacterial disease of acute or chronic course, caused by spirochetes of the genus Leptospira, with zoonotic and cosmopolitan character, which affects humans, wild and domestic animals. It can be transmitted directly by contact with fluids containing leptospires through placental, hematogenous and genital routes and through the act of breastfeeding; or indirectly by contact with an environment contaminated with leptospires. Acquiring knowledge about the infection severity, the geographical distribution, the risk factors, and the circulating strains is of utmost importance to stablish the epidemiology and to improve preventive and diagnostic measures. In this study, recombinant proteins of Leptospira spp. were evaluated as antigens in the development of a rapid immunochromatographic assay based on the type dipsticks a method of diagnosing animal leptospirosis. 11 recombinant proteins were selected as candidate antigens. The purified recombinant proteins were evaluated in the detection of specific antibodies by Western blotting and ELISA. Only the LipL32 protein showed reactivity with positive sera for leptospirosis. Two immunochromatographic tests, using LipL32 protein, have been developed: a type I test using the LipL32 protein conjugated to colloidal gold and another, type III with colloidal gold conjugated to protein A and LipL32 in the test line. In both assays the test lines and the control lines reacted, showing that the methods are working. The type I test performed manually showed satisfactory results for bovine and hyperimmune rabbit sera. The type III test showed reactivity for bovine, horse, rabbit and dog sera. When these two tests were applied in automated machinery, it has not been possible to detect the line test, probably due to the need for further standardization of all method parameters.

Chi Keung Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 219-251). / Chapter CHAPTER I --- LITERATURE REVIEW / Chapter 1 --- The structure of bone --- p.2 / Chapter 1.1. --- The cortical bone --- p.3 / Chapter 1.2. --- The cancellous bone --- p.3 / Chapter 2 --- The composition of bone --- p.3 / Chapter 2.1. --- Bone minerals --- p.4 / Chapter 2.2. --- The organic matrix --- p.4 / Chapter 2.3. --- The bone cells --- p.9 / Chapter 2.3.1. --- The osteoblast and the osteocyte --- p.9 / Chapter 2.3.2. --- The osteoclast --- p.11 / Chapter 3 --- Bone turnover - modelling and remodelling of bone --- p.13 / Chapter 3.1. --- Postulated sequence of bone remodelling --- p.14 / Chapter 4 --- Regulation of bone resorption --- p.16 / Chapter 4.1. --- Role of osteoblast and the lining cell on bone resorption --- p.17 / Chapter 5 --- Regulation of bone formation --- p.19 / Chapter 6 --- Effects of systemic hormones and local factors on bone metabolism --- p.20 / Chapter 6.1. --- Parathyroid hormone --- p.20 / Chapter 6.2. --- "1,25-dihydroxyvitamin D3" --- p.22 / Chapter 6.3. --- Calcitonin --- p.23 / Chapter 6.4. --- Prostaglandins --- p.23 / Chapter 6.5. --- Sex hormones --- p.24 / Chapter 6.6. --- Glucocorticoid --- p.26 / Chapter 6.7. --- Growth hormone --- p.27 / Chapter 6.8. --- Insulin --- p.28 / Chapter 6.9. --- Thyroid hormones --- p.29 / Chapter 6.10. --- Other systemic and local factors --- p.30 / Chapter 7 --- Indices of bone turnover --- p.34 / Chapter 8 --- Non-biochemical indices of bone metabolism --- p.34 / Chapter 8.1. --- Radionuclide bone scan --- p.34 / Chapter 8.2. --- Radiokinetic assessment --- p.35 / Chapter 8.3. --- Bone biopsy --- p.35 / Chapter 8.4. --- Bone densitometry --- p.36 / Chapter 9 --- Biochemical indices of bone metabolism --- p.37 / Chapter 10 --- Biochemical markers of bone formation --- p.38 / Chapter 10.1. --- Alkaline phosphatase --- p.38 / Chapter 10.1.1. --- Role and origin of bone alkaline phosphatase isoenzyme --- p.39 / Chapter 10.1.2. --- Measurement of bone alkaline phosphatase --- p.41 / Chapter 10.1.2.1. --- Heat inactivation --- p.42 / Chapter 10.1.2.2. --- Chemical inactivation --- p.43 / Chapter 10.1.2.3. --- Immunological methods --- p.44 / Chapter 10.1.2.4. --- High performance liquid chromatography --- p.45 / Chapter 10.1.2.5. --- Gel electrophoresis --- p.45 / Chapter 10.1.2.6. --- Isoelectric focusing --- p.47 / Chapter 10.2. --- Osteocalcin --- p.48 / Chapter 10.3. --- Osteonectin --- p.51 / Chapter 10.4. --- Matrix Gla-protein --- p.51 / Chapter 10.5. --- Other non-collagenous proteins --- p.52 / Chapter 10.6. --- Urinary Gla concentration --- p.52 / Chapter 10.7. --- Collagen peptides and extension peptides --- p.54 / Chapter 11 --- Biochemical markers of bone resorption --- p.55 / Chapter 11.1. --- Urine hydroxyproline --- p.55 / Chapter 11.2. --- Pyridinium cross-links --- p.58 / Chapter 11.3. --- Acid phosphatase --- p.60 / Chapter 11.3.1. --- Acid phosphatase isoenzymes --- p.60 / Chapter 11.3.2. --- The band 5 acid phosphatase isoenzyme genetics and characteristics --- p.62 / Chapter 11.3.3. --- Band 5 acid phosphatase as marker of osteoclastic function --- p.64 / Chapter 11.3.4. --- Measurement of osteoclastic acid phosphatase --- p.67 / Chapter 11.3.4.1. --- Specific chemical inhibitor --- p.67 / Chapter 11.3.4.2. --- Electrophoresis --- p.67 / Chapter 11.3.4.3. --- Immunological methods --- p.68 / Chapter 12 --- Problems with current biochemical markers of bone metabolism --- p.68 / Chapter 13 --- Aims of this study --- p.70 / Chapter CHAPTER II --- PURIFICATION OF TARTRATE-RESISTANT ACID PHOSPHATASE AND THE DEVELOPMENT OF AN IMMUNOASSAY FOR IT'S MEASUREMENT / Chapter 1 --- Introduction --- p.72 / Chapter 2 --- Materials and methods --- p.75 / Chapter 2.1. --- Chemicals and reagents --- p.75 / Chapter 2.1.1. --- Apparatus --- p.76 / Chapter 2.2. --- Methods --- p.77 / Chapter 2.2.1. --- Cord serum --- p.77 / Chapter 2.2.2. --- Measurement of tartrate-resistant acid phosphatase activity --- p.77 / Chapter 2.2.3. --- Measurement of protein concentration --- p.80 / Chapter 2.2.4. --- Purification of TRACP from cord plasma --- p.82 / Chapter 2.2.4.1. --- Cation-exchange column chromatography --- p.83 / Chapter 2.2.4.2. --- Gel filtration column chromatography --- p.84 / Chapter 2.2.4.3. --- Concanavalin A-affinity column chromatography --- p.85 / Chapter 2.2.4.4. --- Preparative isoelectric focusing (IEF) --- p.86 / Chapter 2.3. --- Characterisation of purified TRACP --- p.90 / Chapter 2.3.1. --- Polyacrylamide gel electrophoresis (PAGE) --- p.91 / Chapter 2.3.2. --- "Optimum pH, substrate specificity and the effects of potential activators and inhibitors on TRACP activity" --- p.99 / Chapter 2.3.3. --- Amino acid composition of purified TRACP --- p.101 / Chapter 2.4. --- Methods for raising anti-human TRACP antibody and characterisation of the antiserum --- p.102 / Chapter 2.4.1. --- Production of rabbit anti-human TRACP antibody --- p.102 / Chapter 2.4.2. --- Determination of the titre of rabbit anti-human TRACP antibody --- p.103 / Chapter 2.4.3. --- Immunoblotting analyses for cross reactivity study --- p.103 / Chapter 2.4.4. --- Immunohistochemical study for antibody specificity --- p.105 / Chapter 2.4.5. --- Cross reactivity study of the rabbit anti-human TRACP antibody to some tissue preparations --- p.107 / Chapter 2.5. --- Enzyme linked immunosorbent assay for TRACP --- p.109 / Chapter 2.5.1. --- Optimisation and evaluation of the new ELISA method for TRACP --- p.111 / Chapter 3 --- RESULTS --- p.113 / Chapter 3.1. --- "Precision of methods for the determination of protein, TRACP and phosphate." --- p.113 / Chapter 3.2. --- Isolation and purification of TRACP --- p.113 / Chapter 3.2.1. --- Concanavalin A affinity chromatography --- p.120 / Chapter 3.2.2. --- Isoelectric focusing (IEF) --- p.120 / Chapter 3.3. --- Characterisation and homogeneity of purified TRACP --- p.128 / Chapter 3.3.1. --- Characterisation of purified TRACP --- p.128 / Chapter 3.3.2. --- Homogeneity of purified TRACP --- p.132 / Chapter 3.3.3. --- Amino acid composition --- p.136 / Chapter 3.4. --- Characterisation of the rabbit anti-human TRACP antibody --- p.136 / Chapter 3.4.1. --- Antibody specificity - immunoblotting study --- p.139 / Chapter 3.4.2. --- Antibody specificity - cross reactivity with partially purified non-cord plasma TRACP --- p.142 / Chapter 3.4.3. --- Antibody specificity - immunohistochemical study --- p.145 / Chapter 3.5. --- Enzyme linked immunosorbent assay for TRACP --- p.145 / Chapter 3.5.1. --- Optimal concentration of antigen for coating of microtitre plate --- p.145 / Chapter 3.5.2. --- Kinetics of reaction with the primary rabbit anti-human TRACP antibody --- p.149 / Chapter 3.5.3. --- "Precision, recovery and assay range" --- p.149 / Chapter 4 --- DISCUSSION --- p.155 / Chapter 4.1. --- Purification of cord plasma TRACP --- p.155 / Chapter 4.2. --- Characterisation of cord plasma TRACP --- p.158 / Chapter 4.3. --- Characterisation of rabbit anti-human TRACP antibody --- p.163 / Chapter 4.4. --- Enzyme immunoassay for TRACP --- p.165 / Chapter CHAPTER III --- STUDY OF SERUM TRACP IN HEALTHY SUBJECTS AND IN PATIENTS WITH BONE RELATED DISEASES / Chapter 1 --- Introduction --- p.168 / Chapter 2 --- Materials and methods --- p.171 / Chapter 2.1. --- Subjects --- p.171 / Chapter 2.1.1. --- Healthy subjects --- p.171 / Chapter 2.1.2. --- Patients --- p.172 / Chapter 2.1.2.1. --- Post-menopausal women on hormone replacement therapy --- p.172 / Chapter 2.1.2.2. --- Hip fracture patients --- p.173 / Chapter 2.1.2.3. --- Other patients --- p.174 / Chapter 2.3. --- Measurement of other biochemical parameters --- p.175 / Chapter 2.3.1. --- Bone alkaline phosphatase --- p.175 / Chapter 2.3.2. --- "Measurement of urine hydroxyproline, creatinine, calcium, osteocalcin, thyroid hormones and parathyroid hormone" --- p.176 / Chapter 2.4. --- Statistics --- p.178 / Chapter 3 --- RESULTS --- p.179 / Chapter 3.1. --- Healthy subjects --- p.179 / Chapter 3.2. --- Serum TRACP concentration in post-menopausal women before and after hormone replacement therapy --- p.185 / Chapter 3.3. --- TRACP concentration in elderly subjects with hip fractures --- p.189 / Chapter 3.4. --- Serum TRACP concentrations in patients with other bone related diseases --- p.190 / Chapter 3.4.1. --- Hyperthyroidism --- p.194 / Chapter 3.4.2. --- Hyperparathyroidism --- p.198 / Chapter 3.4.3. --- Haemodialysis --- p.201 / Chapter 4 --- DISCUSSION --- p.204 / GENERAL DISCUSSION --- p.216 / REFERENCES --- p.219

The current project work aims to develop a simple and sensitive immunoassay for prostate specific antigen (PSA) by using changes of fluorescence of gold nanoparticles with varying coverage of proteins. In this work we attempted to investigate the changes in optical properties caused by coating the nanoparticles with antibody-antigen complex. We wanted to construct biosensor that utilizes these changes to monitor biological bindings without fluorescent marker.The underlying idea of the project has been to use label free immunoassay to monitor not only presence or absence of antigens in sample solution but also to quantify the antigen concentration, we have tried to develop a simple and time and labour saving method based on a non competitive heterogeneous but label free immunoassay.There are many instrumentation techniques for analysis of changes of optical properties of nanoparticles used in an immunoassay, like absorption spectroscopy, surface plasmon resonance spectroscopy, Raman spectroscopy, time resolved fluorescence and electrochemical techniques [1]. In present work we have investigated whether the adsorption of antibodies onto Au (gold) nanoparticles would change the optical properties of antibodies to an extent sufficient to differentiate them from the free antibodies. We have furthermore investigated whether the subsequent antigen binding to antibodies also induces changes of optical properties sufficient for quantitative analysis. We have chosen to monitor optical properties via measurement of fluorescence because of its sensitivity and selectivity.Our objective here has been not only to investigate spectral changes but also to develop a robust assay protocol, for example with respect to the antibody binding and nanoparticles separation techniques. Two critical steps of the experimental procedure developed here have been (i) the separation of excess prostate specific monoclonal antibodies (PSA10) from the solution containing nanoparticles with adsorbed PSA10 and free PSA10, and the stability of Au-PSA10 conjugates, and (ii) quantification of the binding of PSA to PSA10 covered nanoparticles.We have encountered problems with agglomeration of gold nanoparticles, both naked and with PSA10 conjugates. The naked nanoparticles were “sticky” and bound easily with other materials, for example with agarose beads from the separation columns. Fortunately the coated nanoparticles turned out to be much more inert. This allowed the separation and, simultaneously, acted as a test for antibody coverage. / Uppsatsnivå: D