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In this study, the magnetically-induced vibrations of functionalized magnetic particle suspensions were probed for the development of a novel optical spectroscopy technique. Through this work (1) the frequency-dependence of the faraday rotation in ferrofluids and (2) the extension of this system to elucidating particle size and conformation as an alternative immunossay to costly and labor/time intensive Western Blotting and ELISA has been shown. With its sensitivity and specificity, this method has proven to be a promising multi-functional tool in biosensing, diagnostic, and therapeutic nanomedicine efforts. Due to its ubiquitous nature in all optically-transparent materials, the farady rotation, or circular birefringence, was developed as a robust and sensitive nanoscale biomolecule characterization technique through Brownian relaxation studies of particle suspensions. Current efforts have shown the applicability of this phenomenon in solid, pure liquid, and colloidal samples as well as simultaneous advancements of magnetic nanoparticle research in the magnetometric and magneto-optical regimes. By merging these two fields, a clinically relevant spectroscopy (fd-FRS, Frequency Domain Faraday Rotation Spectroscopy) was developed based on a newly revised model stemming from Debye relazation theory. Through this work, an optical bench with a variable permeability core electromagnet and a frequency-domain lock-in amplifier setup (DC to 20 kHz) have been used to distinguish between Fe3O4-core nanoparticles with functionalization layers of PEG4/PEG8 polymer with future applications involving the Anti-BSA/BSA antibody/antigen couple. Particle concentrations down to 500 nM (magnetic nanoparticles) and 0.01 Volume % (magnetic beads) were studied with diameters ranging from 200 nm to 1μm. currently, the characteristic peak corresponding to the out-of-phase relazation of the suspended particles has been elusive, despite a wide particle size distribution and the use of a balanced photodetector. Future work will involved highly monodisperse samples, faster scan times, and thermal characterization applications of fs-FRS.

Составлен аналитический обзор литературы по иммуноанализу, применению наночастиц магнетита в иммуноанализе. Синтезированы наночастицы магнетита, аминированные наночастицы магнетита, конъюгаты наночастиц магнетита с антителами к вирусу кори. Зарегистрирован прямой аналитический электрохимический сигнал от аминированных наночастиц Fe3O4, конъюгатов наночастиц магнетита с антителами к вирусу кори. Проведен анализ модельных и реальных растворов с использованием схем конкурентного и неконкурентного гетерогенного иммуноанализа. / An analytical review of the literature on immunoassay, application of magnetite nanoparticles in immunoassay is made. Synthesized magnetite nanoparticles, aminated magnetite nanoparticles, magnetite nanoparticle conjugates with antibodies to measles virus. The analysis was performed using competitive and noncompetitive immunoassay schemes. A direct analytical signal from magnetic nanoparticles, aminated nanoparticles, magnetite nanoparticle conjugates with antibodies to measles virus, as well as from a magnetic tag in immunocomplex is registered.

An analytical review of the literature on immunoassay, the mobilization of biocomponents on the surface of the transducer by electrographing was done. Methods for the synthesis and immobilization of a linercium compound (paranitrobenzodiazonium chloride) for the realization of electrographing have been developed. The transducer for immunoassay is selected. The working conditions for carrying out the immunoassay are determined using the example of model systems. Metrological processing of results is carried out. / Составлен аналитический обзор литературы по иммуноанализу, иммобилизации биокомпонентов на поверхность трансдьюсера методом электрографтинга. Разработаны методики синтеза и иммобилизации линкерного соединения (хлорида паранитробензодиазония) для реализации электрографтинга. Выбран трансдьсер для иммуноанализа. Определены рабочие условия проведения иммуноанализа, на примере модельных систем. Проведена метрологическая обработка полученных результатов.

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Diabetes is a significant health burden worldwide. The most common types of diabetes, type 1 and type 2 diabetes are typically characterized by complete insulin deficiency and varying degrees of insulin deficiency, respectively. Glycated hemoglobin (HbA1c), formed when hemoglobin and glucose combine through glycation, can be used to monitor treatment in diabetic patients, and thus prevent future complications of the disease. HbA1c can be measured using point-of-care (POC) instruments, providing rapid results and immediate feedback on HbA1c levels. Measurement with POC instruments can reduce the need for additional visits for blood sampling, thereby lowering costs for both patients and healthcare systems. The main purpose of this study was to verify HbA1c on the POC instruments QuikRead Go and DCA Vantage by comparing the results with the reference method Cobas Pro and by comparing capillary and venous blood samples. The study utilized 30 venous patient samples, including 20 samples already analyzed on Cobas Pro and 10 samples collected venously and capillary from volunteer individuals. The coefficient of variation (CV) for QuikRead Go fell within the quality goal, while DCA Vantage exceeded the goal. The results demonstrated good agreement between capillary blood samples analyzed on POC instruments and venous samples analyzed on Cobas Pro. However, a statistically significant difference was found comparing venous samples analyzed on POC instruments and Cobas Pro. The results suggest that capillary sampling should be used for analysis on POC instruments. Certain limitations of the study should be considered when using QuikRead Go and DCA Vantage in practice.

Affinity-based proteomics offers opportunities for the discovery and validation of disease-associated proteins in human body fluids. This thesis describes the use of antibody-based immunoassays for multiplexed analysis of proteins in human plasma, serum and cerebrospinal fluid (CSF). This high-throughput method was applied with the objective to identify proteins associated to clinical variables. The main work in this thesis was conducted within the diseases of multiple sclerosis and malignant melanoma, as well as mammographic density, a risk factor for breast cancer. The suspension bead array (SBA) technology has been the main method for the work presented in this thesis (Paper I-IV). SBA assays and other affinity proteomic technologies were introduced for protein profiling of sample material obtained from clinical collaborators and biobanks. Perspectives on the validation of antibody selectivity by means of e.g. immuno-capture mass spectrometry are also provided. Paper I describes the development and application of a protocol for multiplexed pro- tein profiling of CSF. The analysis of 340 CSF samples from patients with multiple sclerosis and other neurological disease revealed proteins with potential association to disease progression (GAP43) and inflammation (SERPINA3). Paper II continued on this work with an extended investigation of more than 1,000 clinical samples and included both plasma and CSF collected from the same patients. Comparison of disease subtypes and controls revealed five plasma proteins of potential diagnostic relevance, such as IRF8 and GAP43. The previously reported associations for GAP43 and SERPINA3 in CSF was confirmed. Subsequent immunohistochemical analysis of post-mortem brain tissue revealed differential protein expression in disease affected areas. In Paper III, 150 serum samples from patients with cutaneous malignant melanoma were analyzed. Protein profiles from antibody bead arrays suggested three proteins (RGN, MTHFD1L, STX7) of differential abundance between patients with no disease recurrence and low tumor thickness (T-stage 1 and 2) compared to patients with high tumor thickness (T-stage 3 and 4) and disease recurrence. We observed MTHFD1L expression in tissue of a majority of patients, while expression of STX7 in melanoma tissue had been reported previously. Paper IV describes the analysis of protein in plasma in relation to mammographic breast density (MD), one of the strongest risk factors for the development of breast cancers. More than 1,300 women without prior history of breast cancer were screened. Linear associations to MD in two independent sample sets were found for 11 proteins, which are expressed in the breast and involved in tissue homeostasis, DNA repair, cancer development and/or progression in MD. In conclusion, this thesis describes the use of multiplexed antibody bead arrays for protein profiling of serum, plasma and CSF, and it shortlists disease associated proteins for further validation studies. / <p>QC 20170302</p>

A Doença de Chagas é um dos maiores problemas de saúde pública na América do Sul causando um elevado prejuízo à população. A despeito dos inúmeros esforços para o seu controle, a doença não tem cura e apresenta problemas científicos ainda não esclarecidos. Considerando-se que vários pesquisadores têm usado a radiação ionizante para modificar protozoários ou propriedades imunológicas de biomoléculas, neste trabalho foram estudados aspectos da resposta imunológica induzida em camundongos, resistentes e suscetíveis ao T. cruzi, utilizando formas irradiadas deste parasita. Doses baixas de radiação preservaram a capacidade reprodutiva e de invasão celular. Animais resistentes e suscetíveis, imunizados com os parasitas tratados por radiação, produziram anticorpos específicos. Após o desafio, os animais apresentaram baixa parasitemia, com exceção dos grupos imunizados com parasitas que receberam apenas altas doses de radiação. A seleção de formas tripomastigotas foi obtida irradiando-se os parasitas com baixas doses, o que promoveu aprimoramento da qualidade da resposta imune, a exemplo do que se observa quando da utilização de complemento. Estes dados evidenciam a importância da seleção das formas tripomastigotas para a imunização contra o T. cruzi e apontam a radiação ionizante como alternativa para este fim, uma vez que quando a seleção é feita utilizando-se complemento, depara-se com a dificuldade de sua remoção, colocando em risco o processo de imunização por introduzir substancias estranhas ao organismo. / Chagas\'s disease is one of the major public health problems in South America, promoting high prejudice to the local population. Despite the massive efforts to control it, this disease has no cure and presents puzzling unsolved questions. Considering that many researchers have used ionizing radiation to modify protozoans or biomolecules, we investigated the immunological response aspects of susceptible and resistant mice using irradiated parasites. Low radiation doses preserved the reproductive and invasive capacities of the parasite. Both susceptible and resistant animals, after immunization with irradiated parasites produced specific antibodies. After a challenge, the animals presented low parasitaemia, excepting those immunized with the antigen irradiated with higher doses. Using low radiation doses, we were able to selectively isolate trypomastigotes, leading to an improvement in the quality of the immune response, as previously reported when performing complement system assays. These data highlight the importance of selecting trypomastigote forms for immunization against T. cruz; and point towards ionizing radiation as an alternative to achieve this selection, since when this procedure is performed using complement, the subsequent steps are impaired by the difficulties to remove this component from the system.

Innerhalb dieser Arbeit wurde ein kompetitiver ELISA etabliert und validiert, der eine Quantifizierung von Drogen im humanen Serum gemäß geltender Richtlinien ermöglicht. Der Nachweis erzielte zudem vergleichbare Ergebnisse zur Referenzmethode (Massenspektrometrie). Es wurden neun Drogen (Amphetamin, Methamphetamin, Methylenedioxymethamphetamin (MDMA), Tetrahydrocannabinol (THC), Phencyclidin (PCP), Methadon, Morphin, Kokain und Benzoylecgonin) und insgesamt 33 Antikörper verwendet. Alle Reagenzien durchliefen zunächst ein stringentes Validierungsverfahren. Nach Ausschluss möglicher Kreuzreaktivitäten konnten für drei Drogen (Methadon, MDMA und Benzoylecgonin) jeweils ein Antikörper bestimmt werden, der eine spezifische und sensitive Quantifizierung der Droge ermöglichte. Mit dem anti-MDMA Antikörper war zusätzlich der Nachweis in einem miniaturisierten Format (Microarray) möglich. Die verwendeten Antikörper zeigten keine Kreuzreaktivitäten mit anderen Drogen, Antikörpern, dem Probenmaterial oder anderen Versuchskomponenten. Weiterhin wurde kein Einfluss etwaiger Abbauprodukte im langfristig gelagerten Serum beobachtet. Durch die Ergebnisse wird gezeigt, dass ein immunologischer Nachweis von Drogenmissbrauch in Serum eine verlässliche Ergänzung zu bestehenden Methoden darstellt. Bei einer Übertragung des immunologischen Ansatzes auf ein Microarray ergibt sich zudem die Möglichkeit mehrere Drogen und Serumproben parallel nachzuweisen bzw. zu vermessen. Der höhere Probendurchsatz, der kompakte Versuchsaufbau und der geringere Verbrauch an Material gehört zu den größten Vorteilen dieser Technik. / A competitive ELISA was established, which enables a reliable quantification of serological drug samples according to current guidelines. The method achieved comparable results to the standard method mass spectrometry. In total nine drugs (amphetamine, methamphetamine, 3,4-methylenedioxy-methamphetamine (MDMA), tetrahydrocannabinol (THC), phencyclidine (PCP), methadone, morphine, cocaine and benzoylecgonine) and 33 antibodies were tested. All reagents had to pass through a stringent validation process. After exclusion of cross-reactivities antibodies against three drugs (methadone, MDMA, benzoylecgonine) were validated, which allowed a specific and sensitive quantification. Additionally, by using the anti-MDMA antibody a detection of MDMA in serum on microarray (miniaturized format) was achieved. No cross-reactivities were detected for the used antibodies with other drugs, antibodies, sample material or other assay components. Furthermore, no influence of degradation products in long-stored serum was recorded. The results prove, that immunoassays can be a reliable complement to existing methods. By adapting the immunological method to a microarray, the simultaneous quantification of various drugs and serum samples is enabled. The increased through-put, smaller footprint and the decreased consumption of reagents are some of the biggest advantages of this technique.

O hormônio anti-Mülleriano (AMH) é um marcador de reserva ovariana e função testicular. A aplicação clínica desse hormônio requer padronização adequada de valores de referência, de acordo com o imunoensaio utilizado. Os objetivos deste estudo foram: validar o imunoensaio AMH Gen II (Beckman Coulter Company, TX, USA), estabelecer valores normais de AMH em homens e mulheres saudáveis, avaliar a influência do uso de contraceptivos hormonais, tabagismo e índice de massa corpórea (IMC) sobre os valores de AMH e verificar as concentrações séricas desse hormônio em pacientes com síndrome de Turner (ST), síndrome dos ovários policísticos (SOP) e em meninos com criptorquidismo submetidos ao teste de estímulo com rhCG (gonadotrofina coriônica humana recombinante). A validação do ensaio AMH Gen II foi realizada utilizando protocolo simplificado, conforme as recomendações do Clinical and Laboratory Standards Institute. Para estabelecer valores normais de AMH, 133 mulheres e 135 homens saudáveis foram selecionados prospectivamente. Além disso, 66 pacientes com ST, 29 com SOP e 31 com criptorquidismo foram estudados. A sensibilidade analítica e funcional do ensaio AMH Gen II foram 0,02 e 0,2 ng/mL, respectivamente. Os coeficientes de variação intra e interensaio variaram entre 5,2% - 9,0% e 4,6% - 7,8% em diferentes concentrações, respectivamente. A linearidade, paralelismo e estabilidade das amostras apresentaram percentual de recuperação entre 80% - 120%. O ensaio AMH Gen II correlacionou-se fortemente com o ensaio Immunotech anteriormente utilizado (r = 0,9; p < 0,001). No sexo feminino, os valores séricos de AMH demonstraram declínio progressivo com o aumento da idade (r = -0,4; p < 0,001). Por outro lado, os valores de AMH não diferiram entre usuárias e não usuárias de contraceptivos hormonais, entre tabagistas e não tabagistas, bem como entre mulheres obesas e não obesas. No sexo masculino, a idade não influenciou as concentrações séricas de AMH; no entanto, os valores de AMH foram significativamente reduzidos com o aumento do IMC (r = -0,3, p = 0,008), contudo, esses valores permaneceram dentro do intervalo observado no grupo de indivíduos com peso normal. Todas as pacientes com ST apresentaram valores indetectáveis de AMH. Em contraste, na SOP as concentrações de AMH foram significativamente maiores do que nos controles femininos, mas com 76% de sobreposição entre os dois grupos. Em meninos com criptorquidismo, a concordância entre os valores basais de AMH e a testosterona pósestímulo com rhCG foi de 74%, houve correlação positiva significativa entre os valores basais de AMH e a testosterona pós-estímulo (r = 0,5; p < 0,001). Em conclusão, o ensaio AMH Gen II é confiável para a determinação sérica do AMH. Valores normativos de AMH apresentaram ampla faixa de normalidade em ambos os sexos. Valores reduzidos de AMH em homens obesos encontraram-se dentro da faixa de indivíduos normais. Na ST, o potencial uso do AMH para avaliar a reserva ovariana exige estudos longitudinais. A utilização do AMH como um critério diagnóstico da SOP deve ser associada aos critérios já estabelecidos. Em meninos com criptorquidismo, um AMH normal provê informação útil sobre a função testicular, mas não exclui a necessidade do teste de estímulo com rhCG e pacientes com resultados discordantes necessitam de acompanhamento clínico / The anti-Müllerian hormone (AMH) is a marker of ovarian reserve and testicular function. The clinical application of this hormone requires proper standardization of reference values according to the immunoassay used. The aims of this study were: to validate the AMH Gen II immunoassay (Beckman Coulter Company, TX, USA), to establish reference AMH values in healthy men and women, the influence of hormonal contraceptive use, smoking and body mass index (BMI) on the values of AMH and to check serum concentrations of this hormone in patients with Turner syndrome (TS), polycystic ovary syndrome (PCOS) and in boys with cryptorchidism underwent stimulation test rhCG (recombinant human chorionic gonadotropin).The validation of the AMH Gen II assay was performed using simplified protocol following the recommendations of the Clinical and Laboratory Standards Institute. To establish reference AMH values, 133 healthy women and 135 men were prospectively selected. In addition, 66 patients with TS, 29 with PCOS and 31 with cryptorchidism were studied. The analytical and functional sensitivity of the AMH Gen II assay was 0.02 and 0.2 ng / mL, respectively. Intra and inter-assay coefficients of variation in different concentrations ranged between 5.2-9.0% and 4.6-7.8%, respectively. The linearity, parallelism and stability studies showed recovery % between 80 and 120%. The AMH Gen II assay strongly correlated with the previously used Immunotech assay (r = 0.9, p < 0.001). In females, serum AMH showed a progressive decline with increasing age (r = -0.4, p < 0.001). On the other hand, AMH values did not differ between users and nonusers of hormonal contraceptives, smokers and nonsmokers, obese and non-obese. In males, age did not influence the levels of serum AMH, however, AMH values were significantly reduced with increasing BMI (r = -0.3, p = 0.008), but, these values were within the range observed in the group of subjects with body weight. All TS patients showed undetectable AMH levels. In contrast, in PCOS AMH values were significantly higher than in women controls, but with overlap of 76% between the two groups. In boys with cryptorchidism, the concordance between baseline AMH and testosterone after stimulation with rhCG was 74%, there was a significant positive correlation between baseline AMH values and testosterone values after stimulation (r = 0.5, p < 0.001). In conclusion, AMH Gen II assay is reliable for determining the serum AMH. Reference AMH values showed a wide range in both sexes. Reduced values of AMH in obese men were within the range of normal individuals. In ST, the potential use of AMH to assess ovarian reserve requires longitudinal studies. The use of AMH as a diagnostic criterion of SOP must be based on the association with the criteria already established. In boys with cryptorchidism, a normal AMH provides useful information on testicular function, but does not exclude the need for stimulation test rhCG, patients with discordant results require follow-up