A Multiplexed diagnostic approach for cardiovascular disease biomarkers
[eng] In the course of this thesis, the optimal epitopes for the subsequent polyclonal rabbit antibody production for cTnI and NT-proBNP have been studied and chosen. cTnI and NT-proBNP are the most relevant cardio-specific biomarkers for the diagnosis of cardiovascular diseases. With these antibodi...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/102989 |
| Acceso en línea: | https://hdl.handle.net/2445/102989 http://hdl.handle.net/10803/396304 |
| Access Level: | acceso abierto |
| Palabra clave: | Malalties cardiovasculars Ressonància magnètica nuclear Marcadors bioquímics Diagnòstic Cardiovascular diseases Nuclear magnetic resonance Biochemical markers Diagnosis |
| Sumario: | [eng] In the course of this thesis, the optimal epitopes for the subsequent polyclonal rabbit antibody production for cTnI and NT-proBNP have been studied and chosen. cTnI and NT-proBNP are the most relevant cardio-specific biomarkers for the diagnosis of cardiovascular diseases. With these antibodies, a sandwich ELISA for the detection of cTnI has been developed together with a competitive ELISA for the detection of NT-proBNP, both in a microplate format. It has been observed that cTnI has an extraordinary tendency to non-specifically adsorb itself onto surfaces and other biomolecules. This, with the inability to achieve the required detectability for this biomarker, have been the main problems to face with. Regarding the non-specific adsorption, different additives in the analyte or sample buffer have been evaluated. Thus, 0.15% casein in PBST combined with the use of low adsorption microplates (ImmulonTM 2HB) helps considerably to solve this problem. However, the sensitivity obtained for this assay in aqueous buffer is much lower than that required corresponding to the basal levels of cTnI in the blood. For the NT-proBNP ELISA development, the required limit of detection was achieved after studying various parameters related to heterology and other physicochemical parameters. Moreover, a good accuracy with NT-proBNP fortified plasma samples was obtained. It has been possible to develop a multiplexed microarray for the simultaneous detection of 5 biomarkers (cTnI, NT-proBNP, very important in the process of developing cardiovascular diseases, CRP, Cys C and H-FABP). Once the microarray is biofunctionalized using a spatial encoding with the corresponding bioconjugates or capture antibodies, immunoreagents and other biomarkers can be used in a cocktail. Neither cooperativity phenomena (union of immunoreagents that are in solution in sites where there are other biomarkers immobilized) nor cross-reactivity (recognition of different biomarkers from those for which immunoreagents have been developed) have been observed in any case. Only Lp(a) immunoreagents produced such interferences and therefore they were discarded. It has been highlighted the fact that the biomarkers present in different concentration ranges remains one of the main challenges for the multiplexed diagnosis when simultaneous measurements are desired. In this research, it was impossible to quantify the CRP and Cys C in the same microarray than H-FABP, cTnI and NT-proBNP employing direct samples. Fortunately, the fact of using glass surfaces in which 24 microarrays can be printed, has allowed making these measurements in a simultaneous and parallel way. With the multiplexed microarray, it has been possible to measure samples from patients with different pathologies. The results show that the efficiency of this microarray is much higher than that of analyzers currently used in clinical laboratories, regarding the ability to measure multiple biomarkers in a large number of samples in a short time and the coherence of the results. Thus, the microarray developed in this PhD thesis has been able to measure all the biomarkers from all patient samples, while analyzers only analyzed some of them, depending on the pathology, due to the cost (financial and time). The microarray was then able to detect high levels of CRP from patient samples that were not analyzed in clinical laboratory. Additionally, the microarray results are coherent with those obtained with analyzers for those cases in which measures had been made, and the disease had been diagnosed. The results have been satisfactory even in the case of NT-proBNP, which had not reached the detectability baseline even though it was very close. Unfortunately, it was not possible to measure cTnI levels with the microarray as it was expected according to previous studies done with the same immunoreagents. Thus, we can consider this multiplexed microarray as a semi-quantitative method useful for improving the diagnosis of cardiovascular disease for patients who are at different stages of the disease. Finally, preliminary studies have been realized to implement the multiplexed immunochemical system in a fluorescent optical sensor based on the evanescent wave. This was done with the aim to achieve a POC (point-of-care) device suitable to be used outside hospital premises, adapted to non-specialist users, and facilitate the diagnosis of cardiovascular diseases. Unfortunately, the results obtained point to the need to make greater efforts to increase the detectability of the system, since the LOD values obtained are worse than those achieved with the ELISA or the microarray, far from the basal levels in the case of NT-proBNP. The main problem in this thesis has been to reach the detection limits required by some biomarkers. Although in the literature and in the market there are assays that have achieved it, this fact mainly lies in the signal acquisition method as well as in the intrinsic sensitivity of the instrument addressed to do so. |
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