Abstract At present, diagnostic reagent is the most fruitful and important realm in internal biotech product commercialization. Compared to other biotech medicine with the industrial characteristics of complications such as: high investment, high risk, and long-term research and development; diagnostic reagent, relatively, acquires a lower threshold for technique, a short-term research and development, less investment and a short time for feedback. In addition, people gradually acquire the notions of prophylaxis and health care, as a result, the potentiality of diagnostic reagent comes to the market¡¦s notice. The process of diagnostic reagent development can be divided by the mainstream techniques into four phases including: the technology of chemical test, the technology of enzyme test, the technology of immunoassay test, the technology of nuclease molecule test and biochip. From this, we know that the technology of immunity test is still the mainstream in the market, but there is a trend toward the development of the technology of nuclease molecule immunoassay. General Biologicals Corp. is the first built diagnostic reagent manufacture in our country, and, so far, it is the only diagnostic reagent manufacture that meets with the Department of Health¡¦s CGMP standard. The General Biologicals Corp. is the only internal manufacture that produces ELISA and EIA microplate. However, the General Biologicals Corps has less than 1% of six hundred million, and foreign manufactures have it all. The General Biologicals Corps went public in recent years, and the company has a microplate immunoassay diagnostic reagent factory for over twenty years. Those doctors, examiners and sales clerks who used the immunoassay reagent produced by the General Biologicals Corps are all in high positions in this industry. They know this company and also their reagent kits which have a low sensitivity. More important is that the reagent kits are not improved for all these years. The bad impression of the company makes the customers and collaborators have doubts about the quality, the attitude of the personnel and the sincerity of the company. It results in a trust crisis. In light of the five competitiveness analyses in the executive strategies: dynamic competition of the company in enterprise; S.W.O.T competitiveness analysis; smiling curve and bitter smiling curve; red ocean and blue ocean strategy and, finally, the administration of registering examined medical instrument, to see the integral competitiveness of the company in the industrial competitive environment. Can the General Biologicals Corp. seek league and collaboration in the competitors to seize the internal market, and then the overseas market. By these analyses and advises, to reengeneering the General Biologicals Corp. has a new management on the visible foundation. Key Word¡GImmunity Test, Nuclear Molecular Immuno assay. Five Force, Reengeneering

Semiconductor nanocrystals, otherwise known as Quantum dots (Q dots), are a new type of fluorophore that demonstrates many advantages over conventional organic fluorophores. These advantages offer the opportunity to improve known immunofluorescent methods and immunofluorescent biosensors for rapid and portable bacterial detection in water. The detection of the micro organism Escherichia coli O157:H7 by attenuation of a fluorophore’s signal in water was evaluated alone and in the presence of another bacterial species. A sandwich immunoassay with antibodies adhered to SU-8 as a conventional comparison to our novel attenuation detection was also conducted. The assays were tested for concentration determination as well as investigation for cross reactivity and interference from other bacteria and from partial target cells. In order to immobilize the capture antibodies on SU-8, an existing immobilization self-assembly monolayer (SAM) for glass was modified. The SAM was composed of a silane ((3-Mercaptopropyl) trimethoxysilane (MTS)) and hetero-bifunctional cross linker (N-γ-maleimidobutyryloxy succinimide ester (GMBS)) was utilized in this procedure. The SU-8 surface was activated using various acids baths and oxygenated plasma, and it was determined that the oxygenated plasma yielded the best surface attachment of antibodies. The use of direct fluorophore signal attenuation for detection of the target E. coli resulted in the lowest detectable population of 1x10¹ cfu/mL. It was not specific enough for quantitative assessment of target concentration, but could accurately differentiate between targeted and non-targeted species. The sandwich immunofluorescent detection on SU-8 attained the lowest detectable population of 1x10⁴ cfu/ml. The presence of Klebsiella pneumoniae in solution caused some interference with detection either from cross reactivity or binding site blocking. Partial target cells also caused interference with the detection of the target species, mainly by blocking binding sites so that differences in concentration were not discernable. The signal attenuation not only had a better lowest detectable population but also had less measurement error than the sandwich immunoassay on SU-8 which suffered from non-uniformed surface coverage by the antibodies.

abstract: Cancer claims hundreds of thousands of lives every year in US alone. Finding ways for early detection of cancer onset is crucial for better management and treatment of cancer. Thus, biomarkers especially protein biomarkers, being the functional units which reflect dynamic physiological changes, need to be discovered. Though important, there are only a few approved protein cancer biomarkers till date. To accelerate this process, fast, comprehensive and affordable assays are required which can be applied to large population studies. For this, these assays should be able to comprehensively characterize and explore the molecular diversity of nominally "single" proteins across populations. This information is usually unavailable with commonly used immunoassays such as ELISA (enzyme linked immunosorbent assay) which either ignore protein microheterogeneity, or are confounded by it. To this end, mass spectrometric immuno assays (MSIA) for three different human plasma proteins have been developed. These proteins viz. IGF-1, hemopexin and tetranectin have been found in reported literature to show correlations with many diseases along with several carcinomas. Developed assays were used to extract entire proteins from plasma samples and subsequently analyzed on mass spectrometric platforms. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric techniques where used due to their availability and suitability for the analysis. This resulted in visibility of different structural forms of these proteins showing their structural micro-heterogeneity which is invisible to commonly used immunoassays. These assays are fast, comprehensive and can be applied in large sample studies to analyze proteins for biomarker discovery. / Dissertation/Thesis / M.S. Biochemistry 2012

Cette thèse présente le développement de tests immunologiques innovants, rapides et sensibles combinant des nanoparticules superparamagnétiques (SPN) fonctionnalisées et des micro-aimants : nos immuno-essais magnétiques exploitent les forts gradients de champ magnétique de ces micro-aimants pour capturer les complexes immunologiques liés aux SPN. L’attraction magnétique est souvent utilisée en biotechnologies car elle peut générér des forces capables de capturer des molécules d’intérêt. Les immuno-essais sur billes utilisent habituellement des aimants centi- et millimétriques pour capturer des micro-particules. Réduire la taille des particules magnétiques est très intéressant pour réduire les cinétiques de réactions, tout en diminuant les phénomènes de sédimentation et d’agrégation. Cette réduction d’échelle des particules permet aussi d’augmenter la surface de réaction et ainsi d’augmenter la sensibilité des tests. Cependant les aimants millimétriques génèrent des gradients faibles qui capturent difficilement les SPN, trop mobiles. Les micro-aimants de l’Institut Néel génèrent des forts gradients locaux et ainsi des forces magnétiques importantes. Ces technologies innovantes sont utilisées dans cette thèse pour développer des immuno-essais rapides tirant profit de la réduction d’échelle des particules et des aimants, par rapport aux technologies commerciales.Dans un premier temps, nous avons développé un test immunologique magnétique (MagIA) colorimétrique, comme approche innovante du test ELISA. Nous avons réalisé une preuve de concept pour la détection d’anticorps dirigé contre l’ovalbumine et comparé les résultats avec ceux de tests ELISA. Le test MagIA optimisé présente une limite de détection et une zone dynamique similaires au test ELISA développé avec les mêmes réactifs biologiques. Les micro-aimants fabriqués selon la méthode de micro-magnetic imprinting sont intégrés à bas coût dans les micro-puits des plaques multi-puits ELISA, et permettent la capture efficace des complexes immunologiques couplés aux SPN. La méthode est générique est permet de réaliser des tests ELISA en 30 minutes avec le même équipement.Nous avons ensuite développé un test magnétique immunologique avec une détection fluorescente locale tirant profit des propriétés de capture locale des SPN sur les micro-aimants. Ce test permet la quantification de la molécule d’intérêt en à peine 15 minutes sans étape de lavage. Une preuve de concept réalisée sur la détection de l’anticorps anti-ovalbumine a été réalisée, avec des anticorps de détection fluorescents et des micro-aimants fabriqués selon la méthode de thermo-magnetic patterning. La mesure différentielle entre le signal fluorescent provenant des complexes immunologiques couplés aux SPN localisées sur les micro-aimants, et le signal non spécifique (à l’extérieur des micro-aimants) permet la quantification d’une molécule. Ce test MLFIA (magnetically localized FIA) possède des performances jusqu’à 100 fois meilleures que les tests ELISA standard, pour la détection d’anticorps anti-ovalbumine en PBS. Le test MLFIA a ensuite été transféré à la détection de paramètres cliniques tels que la protéine C réactive, l'ostéopontine, et les sérologies de la toxoplasmose (IgG et IgM). La comparaison des résultats avec des méthodes automatisées a montré d’excellentes corrélations. Le test MLFIA présente plusieurs avantages : il est versatile, compatible avec les milieux biologiques, utilise de faibles volumes et requiert peu d’énergie. Ces résultats ouvrent la voie à une nouvelle génération de tests immunologiques sensibles et nous développons désormais un lecteur miniature pour le diagnostic portable. / This thesis reports the development of innovative, sensitive and fast immunoassays combining functionalized superparamagnetic nanoparticles (SPN) and micro-magnets. Our magnetic immunoassays exploit high gradients generated by micro-magnets to capture immune-complexes captured on SPN. Magnetic attraction is widely used in biotechnology, because it provides long-range forces able to capture molecules of interest. Bead-based immunoassays use common centimetre-scale magnets to attract micro-particles. Those magnets generate low magnetic gradients and struggle to capture superparamagnetic nano-particles, which are too small and mobile to be efficiently trapped. Down-scaling the size of magnetic particles is very interesting since it allows diffusion-based transport to perform faster reactions, while avoiding particle sedimentation and aggregation. Furthermore, it increases the reaction surface, which improves the sensitivity of immunoassays. Thanks to the scaling law effects micro-magnets from Institut Néel generate high local gradients and therefore large magnetic volume forces: we use this innovative technology to develop fast immuno-assays that take advantage of a radical size reduction, compared to commercial technology.We first developed a colorimetric magnetic immunoassay (MagIA) as a new approach to standard ELISA. A proof-of-concept based on colorimetric quantification of anti-ovalbumin antibody in buffer was performed and compared with conventional ELISAs. After optimization, MagIA exhibits a limit of detection and dynamic range similar to ELISAs developed using the same biochemical tools. Micromagnets made by the micro-magnetic imprinting method can be fully integrated in multi-well plates at low cost, allowing the efficient capture of immuno-complexes carried by SPNs. The method is generic and performs magnetic ELISA in 30 min.We then developed a magnetically localized fluorescent immunoassay (MLFIA) exploiting the local capture of SPN on micro-magnets. The differential measurement of fluorescence localized on and besides micro-magnet arrays allows the detection and quantification of a molecule in only 15 minutes without fluid handling. We present a proof of concept based on the detection of monoclonal antibody anti-ovalbumin. Functionalized nanoparticles are incubated with fluorescent detection antibody and the sample containing the molecule to be detected. After a single incubation step, the nanoparticles are captured on micro-magnets made by thermo-magnetic patterning. Fluorescence is then read under a microscope. Differential measurement between the signal from the immunological complex localised on the micro-magnets and the non-specific signal localised besides micro-magnets allows the quantification of mAb anti-OVA. The performance of MLFIA was compared with conventional ELISA and exhibits a limit of detection up to 100 times better for anti-OVA mAb in PBS. For further validation, MLFIA was used to measure clinical parameters: we developed a sandwich assay to detect C-reactive protein, and a serology for Toxoplasma gondii immunoglobulin G and M or osteopontin in human samples. Comparisons with data obtained with routine clinical automatized methods show excellent correlation. Our MLFIA technology presents several key advantages: it is compatible with biological media (serum, plasma), uses small volumes and requires little energy. It also is versatile and thus can be used to detect any antigen or antibody in complex media. We are currently developing a portable prototype for point-of-care diagnostics. The results will open the way to a new generation of sensitive immunological lab-on-chip.

Um dos grandes problemas da engenharia ambiental é a contaminação dos corpos hídricos. Os sanitaristas têm se preocupado com os hormônios sexuais, notadamente os estrógenos, compostos extremamente ativos biologicamente, que têm sido referidos como agentes etiológicos de feminilização e de vários tipos de cânceres. Os estrógenos naturais 17 \'beta\'-estradiol (E2), estriol (E3), estrona (E1) e o sintético 17 \'alfa\'-etinilestradiol (EE2), desenvolvidos para uso médico em terapias de reposição hormonal feminina e métodos contraceptivos, são os que despertam maiores preocupações, pela contínua introdução ao ambiente; hormônios que possuem a melhor conformação reconhecida pelos receptores que resultam respostas máximas, por isso são considerados responsáveis pela maioria dos efeitos disruptores desencadeados pela disposição de efluentes. A mudança de padrões quanto à atividade sexual dos jovens e a preocupação com o planejamento familiar, levaram ao grande consumo de contraceptivos que, através da urina, são levados pela rede coletora aos corpos de água. O indiscriminado uso desses hormônios na bovinocultura, suinocultura, avicultura e aqüicultura são responsáveis por parte considerável desse contaminante nos mananciais. Os hormônios excretados através da urina e fezes e agentes provenientes das indústrias de processamento de alimentos preocupam os sanitaristas porque o lançamento de efluentes in natura ou tratados, são as principais vias de contaminação do ambiente aquático, quer pelo déficit de infra-estrutura em saneamento, quer pela ineficiência tecnológica e/ou operacional na remoção desses compostos nas estações de tratamento de água ou de efluentes. Apesar de possuírem meia-vida relativamente curta, quando comparados a outros compostos orgânicos como praguicidas, os estrógenos naturais são continuamente introduzidos no ambiente, o que lhes confere caráter cumulativo. A proposta desta pesquisa foi verificar a presença de estrógenos na água bruta que chega à Estação de Tratamento de Água, após seu tratamento, em água tratada por osmose reversa e por tecnologia Milli Q (marca registrada). Para verificar e quantificar presença desses hormônios estrógenos em água de abastecimento de São Carlos-SP, foram realizadas exames através de imunoensaio quimiluminescente e radioimunoensaios. Os resultados apontaram que a ETA não possui solução eficiente para a remoção dos analitos de interesse dessa pesquisa, uma vez que na água tratada foram encontrados valores semelhantes aos da água bruta. / One of the major problems of environmental engineering is the water contamination. The sanitary persons have been concerned with the gonadal hormones, notably the estrogen, biologically active compounds extremely, which have been referred to as etiologic agents of feminization and several types of cancers. The natural estrogen 17 \'beta\'-estradiol (E2), estriol (E3), estrone (E1) and synthetic 17 \'alpha\'-ethinylestradiol (EE2), developed for medical use of hormone replacement therapy in women and contraceptive methods, are those that attract larger concerns by the continuous introduction into the environment; hormones that have the best conformation recognized by receptors that result answers maximum, so they are considered responsible for most of the effects disruptors triggered by the wastewater disposal. The change in patterns on the sexual activity of young people and the concern with family planning, led to the large consumption of contraceptives that, in the urine, are led by the distribution net to water. The indiscriminate use of these hormones in cattle, pigs, poultry and aquaculture are responsible for part of this contaminant in the source. The hormones excreted in the urine and feces and agents from the food processing industries in the sanitary concern that the launch of effluents in nature or treated, are the main routes of contamination of the aquatic environment, either by lack of infrastructure, sanitation, or by inefficiency technological and/or operating in the removal of these compounds in the treatment plants, water or effluent. Despite having relatively short stocking-lige when compared to other organics such as pesticides, natural estrogens are continuously released into the environment, which gives them character cumulative. The proposal of this research was to verify the presence of estrogen in the gross water that arrives at the water treatment plant, after the treatment, in water treated by reverse osmosis and by Milli Q technology. To verify and quantify presence of these hormones estrogen in water supply of San Carlos-SP, examinations were conducted through immunoassay chemiluminescent and radioimunoassays. The results showed that the ETA has no efficient solution for removal of interest analytes of this research, because in the treated water were found values similar to crude water.

Um dos grandes problemas da engenharia ambiental é a contaminação dos corpos hídricos. Os sanitaristas têm se preocupado com os hormônios sexuais, notadamente os estrógenos, compostos extremamente ativos biologicamente, que têm sido referidos como agentes etiológicos de feminilização e de vários tipos de cânceres. Os estrógenos naturais 17 \'beta\'-estradiol (E2), estriol (E3), estrona (E1) e o sintético 17 \'alfa\'-etinilestradiol (EE2), desenvolvidos para uso médico em terapias de reposição hormonal feminina e métodos contraceptivos, são os que despertam maiores preocupações, pela contínua introdução ao ambiente; hormônios que possuem a melhor conformação reconhecida pelos receptores que resultam respostas máximas, por isso são considerados responsáveis pela maioria dos efeitos disruptores desencadeados pela disposição de efluentes. A mudança de padrões quanto à atividade sexual dos jovens e a preocupação com o planejamento familiar, levaram ao grande consumo de contraceptivos que, através da urina, são levados pela rede coletora aos corpos de água. O indiscriminado uso desses hormônios na bovinocultura, suinocultura, avicultura e aqüicultura são responsáveis por parte considerável desse contaminante nos mananciais. Os hormônios excretados através da urina e fezes e agentes provenientes das indústrias de processamento de alimentos preocupam os sanitaristas porque o lançamento de efluentes in natura ou tratados, são as principais vias de contaminação do ambiente aquático, quer pelo déficit de infra-estrutura em saneamento, quer pela ineficiência tecnológica e/ou operacional na remoção desses compostos nas estações de tratamento de água ou de efluentes. Apesar de possuírem meia-vida relativamente curta, quando comparados a outros compostos orgânicos como praguicidas, os estrógenos naturais são continuamente introduzidos no ambiente, o que lhes confere caráter cumulativo. A proposta desta pesquisa foi verificar a presença de estrógenos na água bruta que chega à Estação de Tratamento de Água, após seu tratamento, em água tratada por osmose reversa e por tecnologia Milli Q (marca registrada). Para verificar e quantificar presença desses hormônios estrógenos em água de abastecimento de São Carlos-SP, foram realizadas exames através de imunoensaio quimiluminescente e radioimunoensaios. Os resultados apontaram que a ETA não possui solução eficiente para a remoção dos analitos de interesse dessa pesquisa, uma vez que na água tratada foram encontrados valores semelhantes aos da água bruta. / One of the major problems of environmental engineering is the water contamination. The sanitary persons have been concerned with the gonadal hormones, notably the estrogen, biologically active compounds extremely, which have been referred to as etiologic agents of feminization and several types of cancers. The natural estrogen 17 \'beta\'-estradiol (E2), estriol (E3), estrone (E1) and synthetic 17 \'alpha\'-ethinylestradiol (EE2), developed for medical use of hormone replacement therapy in women and contraceptive methods, are those that attract larger concerns by the continuous introduction into the environment; hormones that have the best conformation recognized by receptors that result answers maximum, so they are considered responsible for most of the effects disruptors triggered by the wastewater disposal. The change in patterns on the sexual activity of young people and the concern with family planning, led to the large consumption of contraceptives that, in the urine, are led by the distribution net to water. The indiscriminate use of these hormones in cattle, pigs, poultry and aquaculture are responsible for part of this contaminant in the source. The hormones excreted in the urine and feces and agents from the food processing industries in the sanitary concern that the launch of effluents in nature or treated, are the main routes of contamination of the aquatic environment, either by lack of infrastructure, sanitation, or by inefficiency technological and/or operating in the removal of these compounds in the treatment plants, water or effluent. Despite having relatively short stocking-lige when compared to other organics such as pesticides, natural estrogens are continuously released into the environment, which gives them character cumulative. The proposal of this research was to verify the presence of estrogen in the gross water that arrives at the water treatment plant, after the treatment, in water treated by reverse osmosis and by Milli Q technology. To verify and quantify presence of these hormones estrogen in water supply of San Carlos-SP, examinations were conducted through immunoassay chemiluminescent and radioimunoassays. The results showed that the ETA has no efficient solution for removal of interest analytes of this research, because in the treated water were found values similar to crude water.