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archives@tulane.edu / Groundwater contamination due to the presence of uranium is a subject of concern since chronic exposure to uranium can lead to health problems such as renal failure and cancer. Current standard methods for detection and quantification of uranium in groundwater require expensive instrumentation, laborious sample preparation processes and highly skilled labor to perform. Simple, portable immunosensors can reduce analysis times and costs. Immunosensors take advantage the ability of antibodies to recognize specific molecules. The antibody-antigen binding event can then be read using a quantifiable signal such as color. The success of immunosensors largely depends on the quality of the antibody. In this report, a single chain variable fragment antibody (scFv) was generated from the monoclonal antibody, 12F6 to be used for further studies and re-engineering. The 12F6 antibody binds hexavalent uranium complexed to the chelator, 2,9-dicarboxyl-1,10-phenanthroline (DCP). This scFv was re-engineered in attempt to improve stability as well as adjust it for possible application in a lateral flow device. The full length 12F6 was used to develop a paper-based lateral flow immunoassay device for the detection of uranium in groundwater. Gold nanoparticles were conjugated to the 12F6 antibody to be used as a label. Gold nanoparticles were chosen as a label for this immunoassay due to their biocompatibility and intense plasmonic effect. These immunosensors can be used for rapid testing of groundwater at sites of contamination. This assay could quantify uranium at concentrations below the maximum contaminant level (MCL) for drinking water, 30ppb, or 126nM, as stipulated by the U.S. Environmental Protection Agency (EPA) and the World Health Organization (WHO). / 1 / Grace A. Jairo

A classical radioimmunoassay for hypoxanthine was developed and validated. Hypoxanthine, being a low molecular weight compound (M.W. 136.11) is not immunogenic unless first attached to a macromolecule such as a protein. Derivatives of hypoxanthine were synthesised for this purpose. In addition to the standard preparation of 6-trichloromethyl purine, a derivative of this compound with a 4-carbon spacer arm was prepared, namely purine-6-carboxypropanamide. Hypoxanthine itself showed an unusual degree of stability, having no reactivity towards reagents when considered in either the keto or the enol form. The use of 6-chloropurine, a far more reactive analogue of hypoxanthine, resulted in the synthesis of a novel carboxymethoxylamine derivative, purine-6-carboxymethyl oxime. Four conjugates were prepared using ovalbumin as carrier protein. Hypoxanthine derivatives with a free carboxyl group were conjugated using the mixed anhydride method. 6-trichloromethyl purine was reactive enough to be coupled directly. Hypoxanthine-9-B-D-arabinofuranoside was coupled using the periodate oxidation method for sugar derivatives with vicinal hydroxyl groups. As the hapten was linked via the E-amino groups of lysine residues in each case, the molar derivatisation of each conjugate was calculated by measuring the number of free amino groups in the protein before and after conjugation using 2, 4, 6-trinitrobenzene sulphonic acid. With one of the conjugates ultraviolet spectrophotometric analysis, and calculation of the amount of purine removed during dialysis were also used for comparison and confirmation of the value obtained. The antisera were affinity purified and twelve reagents were compared for their ability to elute anti-hypoxanthine antibodies, whilst retaining immunoreactivity of the eluted fractions. For the determination of hypoxanthine by radioimmunoassay two phase separation systems were investigated, namely chemical precipitation of antigen-antibody complexes using ammonium sulphate, and adsorption of free hypoxanthine using activated charcoal. The antisera were shown to be highly specific for hypoxanthine, with cross-reactivities to six analagous compounds being < 0.1%, and crossreactivity to two further compounds, adenine and allopurinol being 1.9 and 3.2% respectively. Recovery of hypoxanthine added to samples was of the order of 97.3%, and the limit of detection was > 150 nmole/g. Inter-assay coefficient of variation for the data points for the hypoxanthine standard curve was < 10% for hypoxanthine concentrations below 125 nmole/ml. Inter-assay coefficient of variation for samples of fish extract containing hypoxanthine was approximately 12%. Hypoxanthine levels increased with time in samples of trout and whitebait, so that its concentration was indicative of quality, but hypoxanthine levels in liver decreased with length of storage time. Hypoxanthine concentrations in fish samples, as determined by radioimmunoassay, were compared with the values obtained using a well established spectrophotometric method. The correlation coefficient for the two methods was 0.84507 (n = 45) using hypoxanthine solutions extracted from whitebait and 0.93298 (n = 33) for samples of trout muscle, so establishing the radioimmunoassay as a technique for measuring the quality of such foods.

A dosagem do GH no soro é essencial para confirmar ou excluir o seu excesso. Na acromegalia, a ausência de critérios clínicos suficientemente sensíveis para monitorizar o sucesso do tratamento faz com que o GH sérico seja o procedimento de escolha e, para isso, é essencial que a sua dosagem seja realizada de forma confiável, capaz de permitir interpretações uniformes. Vários critérios hormonais têm sido propostos para caracterizar remissão da acromegalia, incluindo níveis séricos de GH randômico inferior a 2,5 μg/l, nadir de GH durante o teste de tolerância oral a glicose inferior a 1,0 μg/l e IGF-I normal para sexo e idade. A importância do tratamento adequado consiste na possibilidade de reverter a mortalidade prematura da acromegalia através da diminuição dos níveis de GH para valores menores que 2,5 μg/l. Com o surgimento de ensaios ultra-sensíveis para medida do GH, tornaram-se necessários critérios mais estritos para determinar cura ou remissão da doença. Nesta revisão, descreveremos aqui as modificações decorrentes da evolução dos ensaios, as conseqüências nos resultados de GH e os pontos de corte propostos na literatura para caracterização da atividade e remissão da acromegalia. / Growth hormone quantification in serum is essential for confirming or ruling out its excess. The absence of clinical criteria sufficiently sensitive to evaluate the treatment success enables GH as the key diagnostic procedure and for that, its measurements must be done in a reliable way and must allow uniform interpretation. Several different biochemical criteria for remission have been suggested in the past, including a random GH measurement less than 2,5 μg/l, mean GH value from a day curve less than 2,5 μg/l, nadir GH value after an oral glucose tolerance test (OGGT) less than 1,0 μg/l and a normal age-related IGFI level. The importance of adequate treatment is highlighted by data indicating that lowering GH levels to less than 2,5 μg/l reverses the premature mortality of acromegaly. With the advances of ultrasensitive assays for GH measurement, stringest remission criteria to determine remission or cure were necessary. In this review, we describe the changes of assay methodology and its consequences in serum GH results and cut off point values to define activity and remission of acromegaly.

A dosagem do GH no soro é essencial para confirmar ou excluir o seu excesso. Na acromegalia, a ausência de critérios clínicos suficientemente sensíveis para monitorizar o sucesso do tratamento faz com que o GH sérico seja o procedimento de escolha e, para isso, é essencial que a sua dosagem seja realizada de forma confiável, capaz de permitir interpretações uniformes. Vários critérios hormonais têm sido propostos para caracterizar remissão da acromegalia, incluindo níveis séricos de GH randômico inferior a 2,5 μg/l, nadir de GH durante o teste de tolerância oral a glicose inferior a 1,0 μg/l e IGF-I normal para sexo e idade. A importância do tratamento adequado consiste na possibilidade de reverter a mortalidade prematura da acromegalia através da diminuição dos níveis de GH para valores menores que 2,5 μg/l. Com o surgimento de ensaios ultra-sensíveis para medida do GH, tornaram-se necessários critérios mais estritos para determinar cura ou remissão da doença. Nesta revisão, descreveremos aqui as modificações decorrentes da evolução dos ensaios, as conseqüências nos resultados de GH e os pontos de corte propostos na literatura para caracterização da atividade e remissão da acromegalia. / Growth hormone quantification in serum is essential for confirming or ruling out its excess. The absence of clinical criteria sufficiently sensitive to evaluate the treatment success enables GH as the key diagnostic procedure and for that, its measurements must be done in a reliable way and must allow uniform interpretation. Several different biochemical criteria for remission have been suggested in the past, including a random GH measurement less than 2,5 μg/l, mean GH value from a day curve less than 2,5 μg/l, nadir GH value after an oral glucose tolerance test (OGGT) less than 1,0 μg/l and a normal age-related IGFI level. The importance of adequate treatment is highlighted by data indicating that lowering GH levels to less than 2,5 μg/l reverses the premature mortality of acromegaly. With the advances of ultrasensitive assays for GH measurement, stringest remission criteria to determine remission or cure were necessary. In this review, we describe the changes of assay methodology and its consequences in serum GH results and cut off point values to define activity and remission of acromegaly.

Made available in DSpace on 2014-06-11T19:29:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-25Bitstream added on 2014-06-13T20:59:11Z : No. of bitstreams: 1 gelamos_jp_me_sjrp.pdf: 2151806 bytes, checksum: 85d6f8deb1daa2021fbe2d398d6c62f7 (MD5) / Universidade Estadual Paulista (UNESP) / Neste trabalho foram desenvolvidos e aprimorados protocolos de conjugação entre nanopartículas do luminóforo Y2O3:Er,Yb, aminofuncionalizadas e a proteína Streptavidina, para atuarem como marcador em imunoensaios. A streptavidina faz parte do sistema de auto-reconhecimento biotina-avidina mais aplicado em ensaios biológicos, sendo que, devido ao seu alto custo, a Albumina Sérica Bovina foi escolhida como proteína de trabalho em substituição à Avidina. Na etapa final, no entanto, utilizou-se a Streptavidina para comparativamente finalizar as discussões. Desta forma, o desenvolvimento do protocolo considerado padrão foi feito aplicando-se o crosslinker homobifuncional glutaraldeído, e a partir deste, estudou-se o comportamento da ligação luminóforo-proteína utilizando-se outros crosslinkers heterobifuncionais, no caso sulfo-N-succinimidil 4-maleimido- butirato sal de sódio (Sulfo-GMBS) e cloridrato de N-(3-dimetilaminopropil)-N'-etilcarbodiimida (EDC). Tal alteração teve como princípio o aperfeiçoamento do protocolo de conjugação, estabelecendo comparativos entres os dados experimentais já obtidos e as possíveis vantagens resultantes dos crosslinkers heterobifuncionais, já que neste caso a possibilidade de ocorrer autoconjugação, ligação cruzada intramolecular e/ou polimerização entre as nanopartículas aminofuncionalizadas pode ser nula. Com relação às nanopartículas, estas, antes da conjugação, foram caracterizadas por espectroscopia de luminescência, microscopia eletrônica de varredura e/ou transmissão, assim como titulação potenciométrica para quantificação dos grupos NH2 após funcionalização. No desenvolvimento e aperfeiçoamento dos protocolos de conjugação todas as etapas foram monitoradas por Espectroscopia de Absorção na Região... / In this work conjugation protocols between the aminofunctionalized nanophosphor Y2O3: Er, Yb, and the protein Streptavidin was developed and improved to be possible applied as markers in immunoassays. The Streptavidin is part of the biotin-avidin self-recognition system applied to most biological assays, and, due to its high cost, Bovine Serum Albumin protein was chosen to be used in most part of the developing protocol as a substitute for avidin. In the final step, however, the Streptavidin was used in comparison to the other results in order to finalize the study. Thus, the development of the standard protocol was done by applying the homobifunctional crosslinker glutaraldehyde, and from this, it was studied the behavior of protein-nanophosphors binding using the heterobifunctional crosslinkers N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and Sulfo-N-succinimidyl 4-maleimidobutyrate sodium salt (Sulfo-GMBS). Such a change had the challenge to improve conjugation protocol, establishing the comparison between experimental data already obtained and the possible advantages due to the use of heterobifunctional crosslinkers, considering that in this case the possibility of occurring self-conjugation, intermolecular crosslinking and/or polymerization between the aminofuctionalized nanoparticles can be null. With respect to the nanoparticles before the conjugation step, they were characterized by luminescence spectroscopy, scanning and/or transmission electron microscopy as well as potentiometric titration for NH2 groups quantification after functionalization. During the development and the improvement of the conjugation protocols all steps were monitored by UV-VIS absorption spectroscopy to verify the behavior of molecular species present before and after the reactions... (Complete abstract click electronic access below)

Orientador: Ana Maria Pires / Banca: Marco Aurélio Cebim / Banca: Eduardo Alves de Almeida / Resumo: Neste trabalho foram desenvolvidos e aprimorados protocolos de conjugação entre nanopartículas do luminóforo Y2O3:Er,Yb, aminofuncionalizadas e a proteína Streptavidina, para atuarem como marcador em imunoensaios. A streptavidina faz parte do sistema de auto-reconhecimento biotina-avidina mais aplicado em ensaios biológicos, sendo que, devido ao seu alto custo, a Albumina Sérica Bovina foi escolhida como proteína de trabalho em substituição à Avidina. Na etapa final, no entanto, utilizou-se a Streptavidina para comparativamente finalizar as discussões. Desta forma, o desenvolvimento do protocolo considerado padrão foi feito aplicando-se o crosslinker homobifuncional glutaraldeído, e a partir deste, estudou-se o comportamento da ligação luminóforo-proteína utilizando-se outros crosslinkers heterobifuncionais, no caso sulfo-N-succinimidil 4-maleimido- butirato sal de sódio (Sulfo-GMBS) e cloridrato de N-(3-dimetilaminopropil)-N'-etilcarbodiimida (EDC). Tal alteração teve como princípio o aperfeiçoamento do protocolo de conjugação, estabelecendo comparativos entres os dados experimentais já obtidos e as possíveis vantagens resultantes dos crosslinkers heterobifuncionais, já que neste caso a possibilidade de ocorrer autoconjugação, ligação cruzada intramolecular e/ou polimerização entre as nanopartículas aminofuncionalizadas pode ser nula. Com relação às nanopartículas, estas, antes da conjugação, foram caracterizadas por espectroscopia de luminescência, microscopia eletrônica de varredura e/ou transmissão, assim como titulação potenciométrica para quantificação dos grupos NH2 após funcionalização. No desenvolvimento e aperfeiçoamento dos protocolos de conjugação todas as etapas foram monitoradas por Espectroscopia de Absorção na Região... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this work conjugation protocols between the aminofunctionalized nanophosphor Y2O3: Er, Yb, and the protein Streptavidin was developed and improved to be possible applied as markers in immunoassays. The Streptavidin is part of the biotin-avidin self-recognition system applied to most biological assays, and, due to its high cost, Bovine Serum Albumin protein was chosen to be used in most part of the developing protocol as a substitute for avidin. In the final step, however, the Streptavidin was used in comparison to the other results in order to finalize the study. Thus, the development of the standard protocol was done by applying the homobifunctional crosslinker glutaraldehyde, and from this, it was studied the behavior of protein-nanophosphors binding using the heterobifunctional crosslinkers N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and Sulfo-N-succinimidyl 4-maleimidobutyrate sodium salt (Sulfo-GMBS). Such a change had the challenge to improve conjugation protocol, establishing the comparison between experimental data already obtained and the possible advantages due to the use of heterobifunctional crosslinkers, considering that in this case the possibility of occurring self-conjugation, intermolecular crosslinking and/or polymerization between the aminofuctionalized nanoparticles can be null. With respect to the nanoparticles before the conjugation step, they were characterized by luminescence spectroscopy, scanning and/or transmission electron microscopy as well as potentiometric titration for NH2 groups quantification after functionalization. During the development and the improvement of the conjugation protocols all steps were monitored by UV-VIS absorption spectroscopy to verify the behavior of molecular species present before and after the reactions... (Complete abstract click electronic access below) / Mestre

A dosagem do GH no soro é essencial para confirmar ou excluir o seu excesso. Na acromegalia, a ausência de critérios clínicos suficientemente sensíveis para monitorizar o sucesso do tratamento faz com que o GH sérico seja o procedimento de escolha e, para isso, é essencial que a sua dosagem seja realizada de forma confiável, capaz de permitir interpretações uniformes. Vários critérios hormonais têm sido propostos para caracterizar remissão da acromegalia, incluindo níveis séricos de GH randômico inferior a 2,5 μg/l, nadir de GH durante o teste de tolerância oral a glicose inferior a 1,0 μg/l e IGF-I normal para sexo e idade. A importância do tratamento adequado consiste na possibilidade de reverter a mortalidade prematura da acromegalia através da diminuição dos níveis de GH para valores menores que 2,5 μg/l. Com o surgimento de ensaios ultra-sensíveis para medida do GH, tornaram-se necessários critérios mais estritos para determinar cura ou remissão da doença. Nesta revisão, descreveremos aqui as modificações decorrentes da evolução dos ensaios, as conseqüências nos resultados de GH e os pontos de corte propostos na literatura para caracterização da atividade e remissão da acromegalia. / Growth hormone quantification in serum is essential for confirming or ruling out its excess. The absence of clinical criteria sufficiently sensitive to evaluate the treatment success enables GH as the key diagnostic procedure and for that, its measurements must be done in a reliable way and must allow uniform interpretation. Several different biochemical criteria for remission have been suggested in the past, including a random GH measurement less than 2,5 μg/l, mean GH value from a day curve less than 2,5 μg/l, nadir GH value after an oral glucose tolerance test (OGGT) less than 1,0 μg/l and a normal age-related IGFI level. The importance of adequate treatment is highlighted by data indicating that lowering GH levels to less than 2,5 μg/l reverses the premature mortality of acromegaly. With the advances of ultrasensitive assays for GH measurement, stringest remission criteria to determine remission or cure were necessary. In this review, we describe the changes of assay methodology and its consequences in serum GH results and cut off point values to define activity and remission of acromegaly.

There is huge academic interest and clinical need associated with the development of biomarker immunoassays where general aims are the generation of highly specific, convenient and sensitive sensing formats. In this project, a powerful electrochemical technique, electrochemical impedance spectroscopy (EIS), is applied in the establishment of powerful biomarker detecting protocols. Firstly, ultrasensitive, label-free and reusable insulin sensors, based on an antibody-PEGylated thiol self-assembly monolayer (PEG thiol SAM) interface, were produced and characterised via Faradaic EIS, presenting a detection limit (LOD) of 1.2 pM, a linear range across four orders of magnitude, and high sensitivity in even 50 % serum. By applying similar surface chemistry, a label-free biosensor, specific for the detection of α-synuclein antibodies, was fabricated. The α-synuclein interfaces used enabled the reliable detecting of this biomarker in patient sample serum. The concentration levels in the control and a patient group were determined to be significantly different, and, significantly, this difference was consistently across two different cohorts. Strikingly, this could potentially underpin an entirely new means of early Parkinson’s disease (PD) diagnosis. Non-Faradaic EIS methods were additionally applied to label-free insulin assays at both PEG thiol SAM and zwitterionic polymer film interfaces. The latter presented not only an exceptionally non-fouling interface, but also one seemingly both highly biocompatible and facilitating enhanced receptor: target binding. Finally, impedance assays, though potent, generally, operate by sampling only one of a limited number of available experimental variables, typically, Rct for Faradaic EIS, or C or Z for non-Faradaic EIS. Work carried out herein also explores the generation and utility of a portfolio of mathematically derived immittance functions all obtained from the same raw data sets. A particular focus was the examination of whether these were capable of increasing assay sensitivity and efficiency above normal impedance treatments.