Academic literature on the topic 'Screening immunoassay'

The aim of the research was to use the principles of immunoassays and fluorescence spectroscopy to detect the presence of 4-amino-N-(4,5- dimethyl-2-pyrimidinyl)benzenesulfonamide, commonly known as sulfamethazine, in food.

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.

[EN] The contamination of water resources with many industrial, agricultural and other chemicals is one of the key environmental problems that humanity is facing nowadays. Despite the fact that they are usually present at very low concentration, they possess a significant risk to aquatic and human life. To address this issue many national and international institutions set different regulations to monitor and control the water quality. Currently, the monitoring of compounds included in official watch lists is conducted by chromatographic and mass spectroscopic methods. These techniques are approved as "gold standards" for analytical quantitation of organic residues in water. Although they are sensitive and reproducible, cannot be used on-site. The need of sampling and centralized laboratory measurements makes not only the overall cost high but lowering the efficiency of the analysis. Therefore, there is an urgent need to develop suitable field methods to facilitate the in situ measurements at a low cost. Biosensors are therefore an alternative technology that can provide sensitive results in a fast and affordable way. This thesis has focused on the development of a biosensor based on immunoassays and compact disk technology, for the multiplex detection of priority water contaminants. As the methods based on the immunorecognition events are challenging in terms of the selectivity and sensitivity, the major part of the thesis was the selection of the immunoreagents, assay form and procedure. For the detection part, gold nanostructures were selected as sensitive tags for signal enhancement. Therefore, different nanoparticles were studied in order to select the optimal size in terms of the signal enhancement, sensitivity and antibody amount used. Also, the assays performances with signal enhancement and without any amplification were evaluated. The best immunoassay was selected for developing the multiplexed assay. Furthermore, an approach to improve the readout sensitivity of microimmunoassays based on used of gold nanoparticles as both capture and detection species was demonstrated. The method is based on the performance of the immunorecognition event in a homogeneous mode and detection part in the heterogeneous format. Finally, representative water samples were analysed to confirm the applicability of the multi-residue assay. The analytical properties have been established for each methodology and the obtained results have been validated by comparison with reference techniques. The investigations carried out in this work, have resulted in new insights in immunoassay technique that could be useful for screening applications.<br>[ES] La contaminación de aguas superficiales causada por plaguicidas y productos industriales es actualmente, uno de los grandes problemas medioambientales. Aunque estas sustancias están presentes a niveles muy bajos, tienen efectos perjudiciales en el medio en general y especialmente en humanos. Por este motivo, diferentes instituciones han regulado los niveles de contaminantes en áreas de controlar de la calidad de las aguas, creando listas prioritarias de sustancias peligrosas y tóxicas para el medio ambiente. Actualmente, la monitorización de los contaminantes incluidos en las listas oficiales se realiza mediante técnicas cromatográficas y espectrometría. Estos métodos analíticos están aprobados como técnicas de referencia para la determinación de residuos orgánicos presentes en aguas naturales. A pesar de ser técnicas fiables, reproducibles y sensibles, los métodos cromatográficos no están exentos de inconvenientes. Este tipo de metodologías requiere una instrumentación costosa y una laboriosa preparación de muestra, que hacen que el análisis sea en general complejo. Por ello, el desarrollo de métodos analíticos alternativos que faciliten hacer medidas in-situ a bajo coste y con gran capacidad de trabajo es de gran utilidad. En este sentido, las técnicas inmunoquímicas tienen un gran potencial analítico ya que son en general sensibles y selectivas, se pueden utilizar en el lugar de la toma de muestra y tienen capacidad multianalito. Esta tesis se ha centrado en el desarrollo de un sistema biosensor, basado en la tecnología de disco compacto, para la detección multianalito de diversos contaminantes prioritarios en aguas naturales. Las limitaciones más críticas para el desarrollo de un biosensor multianalito mediante métodos inmunoquímicos son los relacionados con su sensibilidad y selectividad. Por lo tanto, una parte importante de la tesis se ha centrado en la selección de inmunoreactivos, formato y optimización de diferentes parámetros claves del ensayo. Una estrategia utilizada para aumentar la sensibilidad de los ensayos ha consistido en marcar la inmunoreacción con nanopartículas de oro. Para ello, se ha estudiado diferentes tipos (esféricas y cilíndricas) de distinto tamaño y se han comparado sus prestaciones analíticas (relación señal ruido, sensibilidad etc.) También, se han desarrollado inmunoensayos cuantitativos sin necesidad de amplificación de la señal. Por otro lado, se ha desarrollado una aproximación que hemos denominado "inmunocaptura" basada en el uso de nanopartículas de oro como especie de captura de analitos en disolución y que actúa como marcador de la inmunointeracción que tiene lugar en la fase sólida. Finalmente, se han analizado muestras de agua naturales dopadas con distintos niveles de los analitos objeto de estudio para evaluar la utilidad de las metodologías desarrolladas como herramienta de screening masivo en el área medioambiental. Los resultados obtenidos han sido comparados con los obtenidos mediante las técnicas de referencia. Las investigaciones realizadas han permitido desarrollar nuevos formatos de ensayo y conocimientos inmunoquímicos aplicados a la tecnología de disco compacto, aportando nuevas herramientas de screening que permiten la determinación simultánea de contaminantes en aguas naturales por debajo de las concentraciones establecidas en la normativa europea de calidad de agua.<br>[CA] La contaminació d'aigües superficials causada principalment per plaguicides i altres productes industrials és un dels grans problemes mediambientals actuals. Malgrat que aquestes substàncies estan presents en nivells molt baixos, tenen efectes perjudicials en humans i animals. Per aquest motiu, diferents institucions estatals han regulat els nivells de contaminants en àrees de control de la qualitat de l'aigua, creant llistes prioritàries de substàncies perilloses i tòxiques per al medi ambient. Actualment, la monitorització dels contaminants inclosos en les llistes oficials es realitza mitjançant tècniques cromatogràfiques i d'espectroscòpia de masses. Aquests mètodes analítics estan aprovats com a tècniques de referència per a la determinació de residus orgànics presents en aigües naturals. Malgrat ser tècniques fiables, reproduïbles i sensibles, els mètodes cromatogràfics no estan exempts d'inconvenients. Aquest tipus de metodologies requereix una instrumentació costosa i una laboriosa preparació de mostres que fan que l'anàlisi sigui, en general, complex. Per això, el desenvolupament de mètodes analítics alternatius que facilitin la possibilitat de fer mesures in-situ a baix cost i amb gran capacitat analítica és de gran utilitat. En aquest sentit, les tècniques inmunoquímiques tenen un gran potencial analític ja que són, en general, sensibles i selectives, es poden utilitzar en el lloc de presa de la mostra i tenen capacitat multianalit. Aquesta tesi s'ha centrat en el desenvolupament d'un sistema biosensor, basat en la tecnologia de disc compacte, per a la detecció multianalit de diversos contaminants prioritaris en aigües naturals. Les limitacions més crítiques per al desenvolupament d'un biosensor multianalit mitjançant mètodes inmunoquímics són sensibilitat i selectivitat. Per tant, una part important de la tesi es va centrar en la selecció de inmunoreactius, format i optimització de diferents paràmetres clau de l'assaig. La detecció es va dur a terme mitjançant l'ús de nanopartícules d'or com a marcadors de la inmunointeracció i amplificació de la senyal analítica. S'han estudiat diferents estructures d'or (esferes i cilindres) de diferents tamanys, i s'han comparat les seves prestacions analítiques (relació senyal-soroll, sensibilitat, etc.). També s'han desenvolupat immunoassaigs quantitatius sense necessitat d'amplificació del senyal. Per altra banda, s'ha desenvolupat una aproximació que hem denominat "inmunocaptura", basada en l'ús de nanopartícules d'or com a espècie de captura d'analits en dissolució i que actua com a marcador de la inmunointeracció que té lloc en la fase sòlida. Finalment, s'han analitzat mostres d'aigües naturals dopades amb diferents nivells dels analits objecte d'estudi per avaluar la utilitat de les metodologies desenvolupades com a eina de "screening" massiu en l'àrea mediambiental. Els resultats obtinguts han sigut avaluats per comparació amb els obtinguts mitjançant tècniques de referència. Les investigacions realitzades han permès desenvolupar nous formats d'assaig i coneixements inmunoquímics aplicats a la tecnologia de disc compacte, aportant noves eines de "screening" que permetin la determinació de contaminants en aigües naturals per sota dels límits de concentració establerts per les normes internacionals de la qualitat de l'aigua.<br>Dobosz, PD. (2017). Screening methodologies for the determination of water contaminant residues by compact disk technology [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79548<br>TESIS

Primary aldosteronism (PA) is a common form of secondary hypertension with an international prevalence rates between 5 and 10 %. It is characterized by a high autonomous aldosterone production that causes cardiovascular damage, renin suppression, hypertension, sodium retention, potassium excretion and hypokalemia. The screening of PA is a simple test measuring aldosterone to renin ratio (ARR) with immunoassay method. This test is currently considered as the most reliable screening tool for PA.     The main objective of the study was to evaluate an ELISA-method, for detection of aldosterone and renin in blood plasma, to be used for routine analysis in the laboratory. The second aim was to investigate the effect of refreezing samples, considering that cryoactivation of prorenin might occur.     One hundred blood samples were analysed, in regard to aldosterone and renin, by using two commercial ELISA assays (DRG ELISA from DRG Diagnostics, Germany) on a Dynex DS2 instrument. In addition, the accuracy and precision of the methods were calculated. The effect of refreezing was investigated with a series of eight samples, which were analyzed twice on the same instrument.     Both assays performed well. The resulting data showed good precision and accuracy. The correlation between the original and refreezed samples was good, r = 0.989 and r = 1.0 for aldosterone and renin respectively. Considering that the study only included eight samples, further investigation is recommended.     Evaluation showed that both immunoassays are reliable in diagnostic use and the ELISA-method is suitable to implement in the laboratory for routine analysis.

Large numbers of samples often need to be prepared and analysed in surveys of organic pollutants in the environment, but while the methods commonly used in such surveys can provide abundant detail they are generally costly, time-consuming and require large amounts of resources, so there is a need for simpler techniques. The work underlying this thesis assessed the potential utility of more convenient sample preparation and bioanalytical techniques for rapidly screening various environmental matrices that could be useful complements to higher resolution methods. Initially, the utility of a simplified extraction technique followed by an enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons (PAHs) in authentic (i.e. unspiked) contaminated soils was explored. The results showed that there are relationships between the structure and cross-reactivity among compounds that often co-occur with target PAHs. However, their potential contribution to deviations between estimates of total PAH contents of soils obtained using ELISA and gas chromatography-mass spectrometry (GC-MS) based reference methods were limited. Instead, the cross-reactivity of target PAHs and the failure to extract all of the PAHs prior to the ELISA determinations were the main reasons for these deviations. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were detected in food and feed matrices, as well as in authentic contaminated soils using different bioanalytical techniques – ELISA and two cell-based bioassays: CAFLUX and CALUX (chemically activated fluorescent/luciferase gene expression) assays. In addition, enhanced sample preparation techniques based on accelerated solvent extraction (ASE) were developed. ASE with integrated carbon fractionation (ASE-C) in combination with CAFLUX produced estimates of PCDD and PCDF contents in fish oil and fish meal that agreed well with results obtained using reference methods. Furthermore, results from ELISA and GC-high resolution MS analyses of extracts of PCDD- and PCDF-contaminated soil samples obtained using an adjusted ASE-C technique were strongly correlated. Finally, the thesis reports the first experiments in which the results of CAFLUX, CALUX, and ELISA determinations of PCDDs and PCDFs in extracts of authentic contaminated soil samples were evaluated and compared to those obtained using a reference method. All of the bioanalytical techniques were found to be sufficiently sensitive, selective, and accurate for use in screening in compliance with soil quality assessment criteria. Overall, the improved sample preparation and bioanalytical techniques examined proved to be useful potential complements to conventional methods, enhancing the analytical framework for PAHs, PCDDs, and PCDFs. However, further validation has to be undertaken before they are applied on a large-scale.

The number of monoclonal antibodies approved for therapeutic use has increased rapidlyover the last decade. As a consequence, precise and robust kinetic characterization techniquesare crucial in order to select the best suitable candidates. A kinetic characterization methodwas developed in Gyrolab with automated sample transfers. The characterization wasperformed in solution in a mixing CD, containing an integrated nanoliter mixing chamberwith affinity binding columns. Association rate constants were determined for four anti-TSHantibodies with values ranging from 3x105 M-1s-1 to 10x105 M-1s-1. The antibodies wereranked according to kass. Reproducibility

La recherche de nouvelles réactions est un des enjeux fondamentaux de la chimie organique. En dehors de l’approche classique basée sur la conception d’une réaction en s’appuyant sur les propriétés chimiques des substrats, une nouvelle approche utilisant le criblage systématique de combinaisons aléatoires de fonctions réactives a été récemment adoptée par plusieurs groupes. Cette stratégie nécessite un outil analytique permettant de cribler un très grand nombre de réactions par jour et d’identifier les meilleures combinaisons conduisant à la formation de produits intéressants. Les travaux de thèse présentés dans ce mémoire s’inscrivent dans le contexte de l’utilisation des techniques de dosages immuno-enzymatiques (ELISA) comme outil de criblage pour la recherche de nouvelles réactions de couplage. Dans un premier temps le criblage de 2688 combinaisons de fonctions réactives et de catalyseurs choisies au hasard a été effectué. Ce criblage a permit de mettre en évidence deux nouveaux couplages en présence de sels de cuivre : une réaction entre les thiourées et les phénols conduisant à la formation des isourées et une réaction entre les N-hydroxythiourées et les alcynes conduisant à la formation des thiazole-2-imines. Dans un second temps le criblage de 2816 combinaisons de fonctions sélectionnées, cette fois-ci, de façon rationnelle a été effectué. Ce criblage a visé la découverte de nouvelles cycloadditions [3+2] répondant aux critères de la chimie « click ». Ainsi l’utilisation de dosage immuno-enzymatique a été étendue à l’optimisation des nouvelles réactions découvertes ainsi qu’à l’évaluation de leurs cinétique, chimiosélectivité et biocompatibilité. Près de 3000 tests complémentaires effectuées sur les « hits » issus du criblage primaire ont ainsi permit de mettre en évidence 4 nouvelles réactions de couplage dont une nouvelle réaction « click » : la cycloaddition sydnone-alcyne catalysée au cuivre (CuSAC). Dans la dernière partie de ce manuscrit les études plus détaillées sur la réaction CuSAC ont été effectuées, notamment l’identification de la structure du produit de couplage et l’étendue du champ d’application de cette réaction. Enfin, l’aspect « click » de la réaction CuSAC a été illustré par l’application de cette réaction au marquage d’une protéine<br>Discovery of new reactions is one of the fundamental goals in organic chemistry. In addition to the traditional approach to reaction discovery, consisting in designing a reaction on the basis of known chemical properties of reagents, new approaches based on the screening of random combinations of reactive functions and catalysts have been recently developed. The main prerequisite of this strategy is an analytical tool allowing screening of a big number of reactions per day and identifying combinations leading to the formation of unanticipated products. In the work presented herein a high-throughput immunoassay screening has been used for the discovery of new coupling reactions. In the first part of this work a screening of 2688 combinations of randomly chosen reactive functions and catalysts was carried out. This screening led to the discovery of two copper-promoted coupling reactions: a reaction between thioureas and phenols leading to the formation of isoureas through desulfurization; and a reaction between N-hydroxythioureas and alkynes leading to the formation of thiazole-2-imines. In the second part of the work a screening of 2816 combinations of rationally designed chemical functions and catalysts was carried out. This screening was focused on the discovery of catalytic [3+2] cycloadditions that comply with the standards of “click” chemistry. In this study, the use of immunoassay screening was extended to optimize new reactions and to evaluate their kinetics, chemoselectivity and biocompatibility. Therefore, around 3000 complementary tests were carried out on the hits, identified in the primary screening. This allowed the discovery of 3 new coupling reactions and one new “click” reaction: a copper-catalyzed sydnone-alkyne cycloaddition (CuSAC). The last part of the work was focused on detailed studies of the CuSAC reaction. Identification of the structure of the coupling product and substrate scope of this reaction was carried out. Finally, the applicability of the CuSAC reaction for bioconjugation was demonstrated by an example of protein labeling

碩士<br>長庚大學<br>醫學生物技術暨檢驗學系<br>99<br>According to statistical data of Department of Health, Executive Yuan, R.O.C. (Taiwan), colorectal cancer (CRC) is the third common malignancy in Taiwan. Clinical symptoms are inconspicuous at an early stage of CRC, and it is always combine with metastasis at an advantage stage with 5 years survival rates lower than 10% in patients. Detection of the CRC during an early stage, that can decrease both the incidence and mortality of CRC. A previous study showed (unpublished), Sec61-β, a transmembrane protein that transported proteins pass the endoplasmic reticulum membrane, was overexpressed in 60% of tumor tissues from CRC patients. The western blot data showed 78% sensitivity and 69% specificity. Thus, our study expects to develop an ELISA kit to detect Sec61-β protein in CRC patients’ plasma. First, we prepared rabbit anti-Sec61-beta antibody against full length Sec61-β protein. But it showed low P value (P value= 0.4) in ELISA test. Therefore, We depart Sec61-β protein into two proteins by cloning two cDNA fragments , thus, we expect to obtain two different polyclonal antibodies against different Sec61-β protein site to improve the assay sensitivity and specificity.