Biosensor development has the potential to meet the need for rapid, sensitive, and specific detection of pathogenic bacteria from natural sources. An antibody-based fiber-optic biosensor assay to detect low levels of Vibrio vulnificus in estuarine waters following an enrichment step was developed. The principle of the sensor is based on an immuno-sandwich assay where an anti-V. vulnificus polyclonal capture antibody preparation was first immobilized on a polystyrene fiber-optic waveguide using a biotin-avidin association. The capture antibody is responsible for binding the target cells to the waveguide. Cyanine-5-conjugated anti-V. vulnificus polyclonal antibodies are subsequently allowed to bind to immobilized cells, and detection occurs when a photodetector collects emitted light (670-710 nm) from the fluorophore, which is excited with 635-nm laser light produced by the Analyte 2000 biosensor. Any detection signal greater than a pre-determined threshold signal is considered to be a positive detection event, while any signal lower than the threshold is considered no detection. This immunosensor assay proved highly specific when tested against whole cells and cell extracts from V. cholerae, V. parahaemolyticus, V. alginolyticus, and E. coli. isolates. Following a four hour enrichment in PNCC broth, and in a total of less than seven hours, the assay was able to detect cell extracts from as few as 100 V. vulnificus colony forming units suspended in sterile water. This method holds promise for detection of low numbers V. vulnificus and other autochthonous pathogens in estuarine waters.

Orientador: Guilherme de Paula Nogueira / Banca: Marcelo Vasconcelo Meireles / Banca: Daniel de Jesus Cardoso de Oliveira / Banca: Andréa Fontes Garcia / Resumo: Esse estudo teve como objetivo a padronização de um ensaio imunoenzimático (ELISA) para a determinação das concentrações plasmáticas de IGF-I total, utilizando o sistema de amplificação biotina-estreptavidina peroxidase em um ensaio competitivo. O IGF-I foi extraído da IGFBP, utilizando o tampão glicina acidificado seguido de neutralização do pH com hidróxido de sódio. As microplacas foram sensibilizadas com anti IgG de coelho, e as dosagens realizadas utilizando duas abordagens, um método sem competição (incubação prévia das amostras com o anticorpo anti-h-IGF-I) e outro com competição (adição simultânea de IGF-I biotilinado e amostra). Os melhores resultados foram obtidos utilizando o método competitivo, com a sensibilização da placa com 0,25 μg/poço de IgG anti-coelho, o anticorpo específico na diluição 1:250.000 e 0,06 ng/poço de IGF-I biotinilado. O ensaio in house apresentou, um limite inferior de detecção de 50 ng/mL, uma correlação de 0.945 entre doses quando comparado à uma metodologia comercial. Além disso, após 33 ensaios (1114 amostras) a metodologia apresentou uma boa precisão, com coeficientes de variação inter-ensaio de 12,94% (345,8 ng/mL) para os controles alto e 20,71% (131,6 ng/mL) para o baixo. Dessa forma, conclui-se que a metodologia imunoenzimática para quantificação de IGF-I total utilizando o sistema de amplificação biotina-estreptavidina peroxidase em um ensaio competitivo está estabelecida e apresenta-se como uma ferramenta útil para estudos que vis... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to standardize an enzyme-linked immunosorbent assay (ELISA) to determine plasma concentrations of total IGF-I using the amplification biotin-streptavidin peroxidase system in a competitive assay. The IGF-I was extracted from IGFBPs using the acidified glycine buffer followed by the pH neutralization with sodium hydroxide. The microplates were coated with anti-rabbit IgG, thereafter the measurements were carried out using two approaches, one without competition (prior incubation of samples with the anti-hIGF-I antibody) and another with competition (simultaneous addition of IGF-I and biotinylated sample). The best results were obtained using the competitive method, with the following combination of reagents: microplates were coated with 0.25 µg/well of anti-rabbit IgG, the specific antibody at a dilution of 1:250.000 and 0.06 ng/well of biotinylated IGF-I. The in house methodology showed sensitivity of detection limit of 50 ng/ml, a correlation between doses of 0.945 when compared to a commercial method. In addition, after 33 assays (quantification of 1114 samples) the proposed methodology presented a good precision, with interassay variation coefficients of 12.94% and 20.71% for the high and low controls, respectively. Finally, we concluded that ELISA method for the quantification of total IGF-I using the system biotin-streptavidin-peroxidase amplification in a competitive assay is established and is presented as a useful tool for studies aimed at monitoring ... (Complete abstract click electronic access below) / Doutor

EVALUATION AND DETERMINATION OF THE SENSITIVITY AND SPECIFICITY OF A Treponema pallidum DRIED BLOOD SPOT (DBS) METHOD FOR SEROLOGIC DIAGNOSIS OF SYPHILIS Background: Syphilis is a sexually transmitted infection (STI) caused by Treponema pallidum subspecies pallidum. Syphilis is known as the "great imitator" due to the similarity of clinical signs and symptoms to other infectious diseases. The primary diagnosis of syphilis relies on clinical findings, including the examination of treponemal lesions, and/or serologic tests. Serologic tests are divided into nontreponemal and treponemal tests. Nontreponemal tests are useful for screening, while treponemal tests are used as confirmatory tests. Methods: A total of 200 serum and DBS specimens collected from patients at the Los Angeles Municipal Sexually Transmitted Disease Clinics were tested by the DBS and enzyme immunoassay (EIA) methods. These samples were sent to the Syphilis Diagnostics Laboratory, Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia for testing. Samples were blindly evaluated by the TREP-SPOTTM DBS and the TREP- SURETM EIA methods for the detection of anti-treponemal IgG- and IgM-class antibodies. Results: The sensitivity of the DBS method was 83% (95% CI, 73.89 - 89.50) and specificity was 100% (95% CI, 95.39 - 100)). The positive predictive value and negative predictive values were 100% (95% CI, 94.48 - 100) and 85% (95% CI, 77.43 - 91.0), respectively. The efficiency of the DBS method was 91.5%. The kappa value for the agreement between the DBS method and EIA assay was 0.83 (95% CI, 0.754 - 0.906). The correlation coefficient (r2) between the anti-treponemal antibody assay results obtained from DBS and serum samples was 0.94. Conclusion: DBS is an optimal choice to be used as a screening tool for the detection of anti-treponemal antibodies for the diagnosis of syphilis. The detection of anti-treponemal antibodies (TREP-SPOTTM DBS EIA) compared favorably to the results of serum-base assay (TREP-SURETM EIA), with an overall concordance of 91.5%. Dried blood spots are technically easier to obtain and are suitable blood samples for primary health care centers.

Le diagnostic précoce de l'hypothyroïdie permet d'initier le traitement au plus tôt et ainsi de préserver la santé du patient. Le bénéfice du traitement de l'hypothyroïdie franche a été depuis longtemps établi, mais les critères de prise en charge des patients en hypothyroïdie fruste sont encore difficiles à définir. En effet, les symptômes ne sont pas toujours présents et leur appréciation est subjective. Afin d'établir le diagnostic et la prise en charge, le médecin s'appuie sur le dosage de la thyréostimuline (TSH) dans le sang, qui peut éventuellement être complété par le dosage des hormones thyroïdiennes. Le dosage de la TSH, très sensible, peut présenter sur un même échantillon sanguin d'importantes variations qui rendent d'autant plus difficiles la décision du médecin et le suivi du patient. Le polymorphisme naturel de la TSH peut expliquer en partie ces variations. La TSH appartient en effet à la famille des hormones glycoprotéiques et sa glycosylation peut constituer jusqu'à 30% de son poids. Dans le cas de l'hypothyroïdie en particulier, ces glycanes sont modifiés et présentent une plus grande quantité d'acides sialiques terminaux. Ainsi, certaines variations entre les dosages de la TSH, qui freinent actuellement leur harmonisation, peuvent être dues à des différences de reconnaissance de glycoformes par les anticorps utilisés dans les dosages. Dans ce contexte, l'objectif de de ces travaux était de contribuer à la construction de dosages plus performants que ceux actuellement utilisés dans le diagnostic de l'hypothyroïdie. Un nouveau calibrateur recombinant sialylé plus proche de la TSH circulante dans l'hypothyroïdie a alors été produit. De nouvelles associations d'anticorps monoclonaux ont été utilisées pour construire des dosages. Les nouveaux dosages sélectionnés ont ensuite été calibrés avec la TSH sialylée produite et le calibrateur de référence international. Ils ont alors servi à doser plusieurs séries de sérums de patients. Ces travaux ont donc validé l'utilisation d'un nouveau calibrateur d'origine recombinante pour les dosages de la TSH, ce qui devrait à l'harmonisation des dosages existants. / If iodine deficiency is the first cause of low thyroid hormone levels in the world, there are also other etiologies to thyroid disorders. Diagnosis of those allow an early treatment to preserve patient's health. Although there is a general agreement concerning treatment of overt hypothyroidism, treatment of subclinical hypothyroidism is still under debate. In these cases, symptoms are, by definition, not always present. In order to establish diagnosis, the clinicians rely on the measurement of circulating thyroid stimulating hormone (TSH, potentially completed with thyroid hormones measurement). TSH assays are now very sensitive, but can present important between assays variations. The diagnosis and follow up of the patient are consequently complicated. Natural polymorphism of TSH can explain a part of this variability. TSH belongs to the glycoprotein hormones family and its glycans can count for more than 30% of its weight. In hypothyroidism, these glycans are subject of modulation and present higher levels of terminal sialylation. Variation in immuno-assays can be explained by these modifications of sialylation if recognition by antibodies used in immuno-assays is glycosylation dependent. In this context, the aim of this work was to contribute to the construction of new immuno-assays, more reliable in the early diagnosis of subclinical hypothyroidism. During this thesis a new recombinant standard closer to circulating TSH was produced. The total level of sialylation was higher and better mimic the circulating forms in hypothyroidism. In order to select the best antibodies associations in immuno-assays, new antibodies were obtained and associated with commercially available antibodies. New immuno assays improvement is based on the following two approaches: the first one is the use of a new standard which presents glycoformes closer to the circulating TSH and the second one consists in an appropriate selection of antibodies involved in the assays. The new assays were used to measure TSH concentration in blood samples. These studies associated with validation steps allow us to select four assays and constitute a proof of concept for the use of a new sialylated recombinant standard for TSH assays. This can contribute to the needed harmonization of TSH assays.

There is no rapid diagnostic technique at medical examiners' offices to determine if a decedent is infected with Mycobacterium tuberculosis. Present diagnostic testing requires at least one month for results. A biosensor-based sandwich immunoassay for the detection of M. tuberculosis was developed in this study. M. tuberculosis polyclonal antibody was used for target antigen capture and detection in the immunoassay. Live attenuated M. tuberculosis (ATCC 25177) suspended in phosphate-buffered saline with 0.1% Tween 20 was used as the antigen in the detection assay. The Analyte 2000 was the initial biosensor platform. Initial testing was of Freund's adjuvant complete. M. tuberculosis was detected 50% of the time at 1,000,000 CFU/ml and 100% of the time at 10,000,000 CFU/ml and 100,000,000 CFU/ml. Live attenuated M. tuberculosis was also tested using the Analyte 2000 biosensor. Detection was obtained 87.5% of the time at 1,000,000 CFU/ml and 100% of the time at 10,00 0,000 CFU/ml and 100,000,000 CFU/ml. The RAPTOR, an automated, portable instrument, was then tested as the fiber optic biosensor platform. Positive biosensor detection was obtained 75% of the time at cell concentrations of 1,000,000 CFU/ml, 95% of the time at 10,000,000 CFU/ml, and 99% of the time at 100,000,000 CFU/ml. Live attenuated M. tuberculosis suspended in PBST and seeded into decedent lung tissue was tested using the RAPTOR. Positive detection was obtained 21% of the time at cell concentrations of 1,000,000 CFU/ml, 86% of the time at 10,000,000 CFU/ml and 100% of the time at 100,000,000 CFU/ml. Antibody specificity studies using ELISA were performed to determine the anti-M. tuberculosis antibody's cross reactivity with microorganisms other than M. tuberculosis. M. tuberculosis actively growing in the lung of an individual is found at levels of 10,000,000 to 1,000,000,000 CFU in the lesions of the lung. This study determined that the RAPTOR biosensor assay was capable of detecting the presence of M. tuberculosis in lung tissue homogenate within three hours when the concentration of M. tuberculosis was 10,000,000 to 1,000,000,000 CFU/ml.

The use of smartphone for the detection of biological constituents is becoming a useful tool as a point-of-care (POC) device and diagnostics. When combined with microfluidic paper analytic devices (μPAD) and particle immunoassays, we have the ability to detect bacterial pathogens with sensitivity and specificity. Environmental conditions as well as variability in smartphone imaging and the cellulose in paper microfluidics however can sometimes easily interfere with the detection of small signal changes. Combining this issue with the detection of pathogens in blood (our model biological sample of interest) becomes difficult with such a platform because of the complexity of the sample matrix. However, in this research we take a novel approach at utilizing polystyrene’s auto-fluorescence and the high energy of UV LEDs in a particle immunoassay in order to increase our signal change. We first characterized how the smartphone actually responds to UV light (275-385 nm) with respect to the RGB components in its images. We were then able to determine a favorable response using the 385 nm UV LED. The detection of green fluorescence by polystyrene particles was possible by analyzing the smartphone’s image in the green channel. There was a significant difference in signal change with blood samples including polystyrene versus just blood samples with a normalized signal intensity change of 2.5 (150%). The detection of polystyrene fluorescence was translated into a field deployable prototype, where preliminary trials showed promising results in detecting Escherichia coli in blood with a current limit of detection of 50 CFU/ml. With further experimentation and optimization the limit of detection could be improved to 10 CFU/mL, making it a very useful tool in the detection of blood borne pathogens to prevent complications with onset bacteremia and the more serious cases of sepsis. This assay platform could provide an easy to use solution with detection in a short time (assay time of 1 min) compared to the lengthy blood culture monitoring or biomarker detection.

Immunoassays for 2-methylisoborneol (MIB) and monensin in water were developed, devised and tested to see if the sensitivity could be established and improved. MIB and monensin are hydrophobic haptens with molecular weights of 168 and 671 Da, respectively. Rabbits were immunized with (-) camphor-BSA and (-) borneol-BSA for the production of polyclonal antibodies (pAbs) to MIB. Monoclonal antibodies (mAbs) were produced in Mus musculus using (-) camphor-BSA as immunogen. (+) Bornylamine-thyroglobulin (TG) and MIB-TG were synthesized and used as plate coatings. For the monensin immunoassay, monensin was conjugated to BSA and OVA for immunogen and plate coating, respectively. Several physical parameters that affect the sensitivity of immunoassays including pre-incubation of antibody and antigen, incubation time and temperature, detergent, organic solvents, and ionic strength were evaluated. Improvement of immunoassay sensitivity was also performed by reducing the concentrations of coating antigen and antibodies and using alternative reporter systems such as chemiluminescence (CICL-ELISA), tyramide signal amplification (TSA) and biotin-streptavidin. Different assay formats, i.e., competitive indirect and competitive direct were also compared. Usability of both pAb-based immunoassays for MIB and monensin was evaluated in fortified water samples. A polyclonal-based (pAb) ELISA for MIB had a detection limit of 4.8 ng mL-1 and an IC50 of 105 ng mL-1. Rabbits immunized with (-) camphor-BSA showed a higher immune response than rabbits immunized with (-) borneol-BSA. One clone (i.e., 4F11) of fourteen characterized clones was used to create the monoclonal antibody (mAb)-based ELISA, which had an IC50 of 100.2 ng mL-1 and an LOD of 1.9 ng mL-1. The pAb- and mAb-based CI-ELISA were not specific to MIB alone and cross reacted with camphor and camphor-like compounds. Meanwhile, a pAb-based ELISA for monensin produced a detection limit of 0.1 ng mL-1 and had an IC50 of 1.056-1.090 ng mL-1 with high specificity to monensin. Other reporter systems did not improve the sensitivity of the immunoassays significantly. MIB and monensin polyclonal-based assays showed good correlation to analytical instrumental methods (i.e., GC-MS and LC-MS) in fortified water samples. With a detection limit of ca. 5 ng mL-1 and 0.1 ng mL-1 for MIB and monensin, respectively, both polyclonal-based assays can be used for detection of these analytes in water from different sources and employed as screening tools to complement GC/HPLC-MS instrument methods.

No description available.

The aim of this study was to develop a highly simplified, sensitive and specific malarial diagnostic test at the lowest possible cost. Initial work and optimisation of procedures was achieved with chicken antibodies by covalently attaching commercially available dye to them. Chicken antibodies were easily isolated from egg yolk and dye is cheap, easily visible and requires no equipment for identification of results. A dipstick dye-immunoassay was developed with nitrocellulose as the capture phase. The dye-immunoassay is an alternative to the traditional enzyme linked immunosorbent assay (ELISA) technique, which employs the use of an enzyme-substrate reaction. Numerous dyes were investigated and included Reactive black 5, trypan blue, Cibacron Blue, Congo red, Acid-black 2, dianix blue, dianix red, para-nitroaniline and primulin. Most of these dyes have dark colours which are essential for good contrast on nitrocellulose and in a microtitre plate. Some dyes contain amino (NH2) groups, which are targeted in a covalent linking step and attached to the lysine residues on antibody molecules or to the carbohydrate groups on antibody molecules. Attachment of dye molecules to antibodies with glutaraldehyde was the chief coupling method explored and conditions were optimized in this study. Unbound dye was removed by dialysis. Reactive black 5 is sensitive down to 50 nanograms of antigen on nitrocellulose. A second covalent coupling method was investigated by means of attaching dye to the carbohydrate moieties on the antibody. Reactive black 5 was sensitive down to 50 nanograms of antigen. The carbohydrate method appears to be more sensitive than the glutaraldehyde method at lower antibody concentrations. Primulin, a yellow dye, was similarly investigated. This dye does not have a dark colour initially, but can be diazotized to change its colour to orange or purple. It also fluoresces under ultra-violet light. This dye was sensitive down to 500 nanograms of antigen with both the glutaraldehyde and carbohydrate coupling techniques. A dye-linked immunosorbent assay (D-LlSA) protocol for direct antigen detection has been developed whereby the dye-antibody solution (dianix blue dye) acts as the primary antibody and substrate respectively. Sensitivity levels compare with traditional ELISAs. Dianix blue is sensitive down to 25 nanograms of antigen in a microtitre plate. Unique protein staining abilities of the dyes used in this study were indicated by staining IgY in electrophoretic gels. Acid-black 2 indicated better protein staining abilities than that of Coomassie brilliant blue. Evidence shows that dye was successfully covalently attached to antibodies and that antigen detection is possible by visualising the dye developed spots. Although malarial antibodies were not used, all procedures with chicken antibodies were optimised. Highly simplified, sensitive and specific diagnostic tests were developed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

Surveillance of domestic poultry flocks for antibodies against avian influenza and Newcastle disease to detect and differentiate between these diseases is very important. The ability to determine if the detected influenza virus antibodies belong to one of the reportable H5 or H7 subtypes is imperative. These two major viruses are continually responsible for economic loss in poultry industries all over the world. Current serological methods of detection are an effective means of detecting antibody responses to these viruses, however continually investigating improved methods of surveillance is important. Development of a serological assay using Luminex technology which involves the use of recombinantly generated influenza A nucleoprotein, hemagglutinin H5, hemagglutinin H7, and Newcastle disease nucleocapsid proteins bound to Magplex beads allowed for the simultaneous detection of antibodies against these proteins that matches the efficiency of past methods while maintaining high levels of specificity and overall accuracy. Assay development took the form of two connected projects beginning with construction of an assay that operated in duplex, detecting antibodies against influenza nucleoprotein (AIV-NP) and Newcastle disease nucleocapsid protein (APMV-1-NC). Once optimized, the second half of development involved expansion of the assay to include detection of H5 (AIV-H5) and H7 (AIV-H7) subtypes, as well as the addition of internal assay quality controls to monitor assay performance over time. Assay thresholds and overall performance of both of these functional assays were evaluated using large quantities of field and experimental sera from chickens and turkeys to maximize specificity and overall accuracy.