Immunoassays are widely used for detection of antigens in a wide range of applications including assays in pharmaceutical development. Immunoassays are continuously improved in many aspects including automatization, miniaturization and extending the dynamic range. The need to measure low abundance molecules are challenging and the need to improve the sensitivity is desired. The Gyrolab technology is a miniaturized immunoassay performed in an automated system covering a broad concentration range. In order to  extend the sensitivity, the technology is combined with Binding Oligo Ladder Detection (BOLD) amplification. The technology behind BOLD or Exazym ® utilizes a DNA primer, a polymerase, and a template (RNA) to generate a ladder-like modified DNA strand. Antibodies with affinity for the polymerized DNA:RNA hybrid strand (duplex) conjugated with reporter molecules are introduced to the system, resulting in an increased number of signal-generating molecules associated with each bound analyte molecule. In this thesis, the development of an ultra-sensitive immunoassay is pursued by applying Exazym ® add-on reagents to the Gyrolab platform, comparing performance with the standard Gyrolab sandwich assay and other commercially available high-performing TNF-α assays. The work includes characterization of a wide range of reaction variables involved in the BOLD signal amplification process including hybridization, polymerization, and detection of a synthetic oligonucleotide. The breakthrough involves the introduction of Allophycocyanin (APC) as a fluorescent conjugate, significantly improving sensitivity and signal-to-noise ratios. The BOLD amplified sensitivity for the TNF-α assay approaches levels seen in ultra-sensitive biomarker assays like Erenna ® and Simoa®. Exazym® technology on the Gyrolab platform allows highly sensitive biomarker assays with minimal sample volume and a 1–2-hour run-time. The study marks substantial progress in achieving ultra-sensitive biomarker assays on the Gyrolab platform through BOLD signal amplification. The use of APC-conjugated detection reagents holds promise for future optimization studies.

Made available in DSpace on 2017-07-21T14:35:54Z (GMT). No. of bitstreams: 1 Daniel da Silveira.pdf: 4620671 bytes, checksum: 940d0c2a58ed2267604a5ec3cb5752ba (MD5) Previous issue date: 2016-02-16 / Intracranial hypertension causes several complications when installed, it can severely affect the individual afflicted with permanent sequelae, besides the risk of death. In the case of pregnant women, the risk is even greater due to the high complexity involved in physiological pregnancy period. In this context, the evaluation of intracranial pressure (ICP) could provide important information that can detect these problems and thereby prevent these complications. However, the invasive method that this procedure is performed makes the use of this methodology very limited. It’s necessary the use of a method that is capable of generating this information while providing security to the patient, not generating complications such as infections or injuries resulting from its invasive form. The aim of this study was to evaluate a new technology for noninvasively ICP measuring in pregnant women in a municipal health center in Ponta Grossa, relating it to the clinical and laboratory data obtained at the time of the procedure. It was observed that low and high-risk pregnant women showed a high in ICP. There were also significant differences between groups for laboratory parameters, such as serum levels of alkaline phosphatase and ultra-sensitive C-reactive protein (hs-CRP). The existence of changes in the ICP and its relationship with clinical and laboratory data in the population studied, introduces in the monitoring of pregnant women, a parameter with unknown data due to technical limitations, which allows to identify changes even in the absence of symptoms and to prevent complications related to elevation in intracranial pressure. / A hipertensão intracraniana promove diversas complicações quando instalada, podendo afetar gravemente o indivíduo acometido com sequelas permanentes, além do risco de óbito. Em se tratando de gestantes, o risco é maior ainda, devido à alta complexidade fisiológica envolvida no período gestacional. Nesse contexto, a avaliação da pressão intracraniana (PIC) pode fornecer informações importantes capazes de detectar esses problemas e com isso evitar essas complicações. No entanto, a forma invasiva como é realizado esse procedimento faz com que a utilização dessa metodologia seja bastante limitada. O uso de um método que seja capaz de gerar essas informações e ao mesmo tempo dar segurança ao paciente, não gerando complicações como infecções ou lesões decorrentes de sua forma invasiva, se faz necessário. O objetivo desse trabalho foi avaliar uma nova tecnologia para aferição da PIC de forma não invasiva em gestantes num centro municipal de saúde em Ponta Grossa, relacionando-a a dados clínicos e laboratoriais obtidos no momento da realização do procedimento. Observou-se que gestantes de baixo e alto risco apresentaram PIC elevada. Foram, ainda, encontradas diferenças significativas entre os grupos para os parâmetros laboratoriais, tais como nos níveis séricos de fosfatase alcalina e proteína C reativa -ultrassensível (PCR-us). A detecção da existência de alterações na PIC e sua relação com dados clínicos e laboratoriais, na população estudada, introduz no acompanhamento às gestantes, um parâmetro, com dados até então desconhecidos devido a limitações técnicas, que permite identificar alterações mesmo na ausência de sintomatologia e prevenir complicações relacionadas à elevação da pressão intracraniana.

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Immunoassays are analytical methods that use the highly specific binding of antibodies in order to detect and quantify an analyte. The technique has become a staple in modern biopharmaceutical research and diagnostics, however the measurement of biomarkers like dysregulated cytokines require ultra-sensitive immunoassays that can detect molecules at sub pg/mL concentrations. One such method is the Exazym® signal amplification. Based on a method called Binding Oligo Ladder Detection (BOLD), it is a set of add-on reagents where a primer is conjugated to a detection antibody which is then combined with a template, polymerase and modified DNA nucleotides to generate a oligonucleotide ladder that is detected with a secondary detection antibody; this amplifies the signal by a factor of 10-100 in an existing immunoassay.  By applying this method to the Gyrolab® microfluidic immunoassay system, a sensitivity increase of 880x-1800x was achieved between a pre-synthesised BOLD product and the polymerised BOLD product. Several key factors for successful polymerisation in the microfluidic system were identified: adding the template separately before the polymerase and using a buffer with low ionic strength for the secondary detection antibody. Applying the BOLD amplification to an existing Gyrolab TNF-α assay only resulted in similar sensitivity as previous methods however. This report demonstrates that BOLD amplification can be successfully performed in a flow-through format on miniaturized affinity columns in the Gyrolab system to increase the sensitivity by orders of magnitude, where both the immunoassay and the amplification steps are automated in the system. However, further optimisation is needed for application in biomarker assays.

第23回名古屋大学年代測定総合研究センターシンポジウム平成22(2010)年度報告