A Multiplexed diagnostic approach for cardiovascular disease biomarkers

Abstract

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.

En el transcurs d'aquesta tesi s'han escollit els epítops òptims per a la conseqüent producció d'anticossos policlonals de conill per a cTnl i NT-proBNP, dos dels biomarcadors més cardio-específics i rellevants pel diagnòstic de malalties cardiovasculars. Amb aquests anticossos s'ha desenvolupat un ELISA sandvitx per a la detecció de cTnl i un ELISA competitiu per a la detecció de NT-proBNP, tots dos en format de microplaca.

S'ha observat que la cTnl te una extraordinària tendència a adsorbir-se de forma inespecífica a superfícies i també a altres biomolècules. Pel que fa a l'adsorció inespecífica s'han avaluat diferents additius en el tampó de la mostra o analit veient-se que la caseïna al 0,15% en PBST combinat amb l'ús de microplaques de baixa adsorció (ImmulonTM 2HB) ajuda considerablement a solucionar aquest problema. Tot i això, la sensibilitat obtinguda per aquest assaig en tampó aquós és molt inferior a la requerida corresponent als nivells basals d'aquest analit a la sang.

En el desenvolupament de l'ELISA per NT-proBNP, després d'estudiar diferents paràmetres relacionats amb l'heterologia i altres paràmetres físico-químics, s'ha aconseguit assolir el límit de detecció necessari obtenint una bona exactitud amb mostres de plasma fortificades amb l'analit en qüestió.

Ha estat possible desenvolupar un microarray multiplexat per a la detecció de 5 biomarcardors (cTnl, NT-proBNP, CRP, Cys C i H-FABP). Un cop biofuncionalitzat el microarray amb els corresponents bioconjugats o anticossos de captura, la resta d'immunoreactius i biomarcadors poden ser utilitzats en forma de còctel sense que en cap cas s'hagin observat fenòmens de cooperativitat ni de reactivitat creuada. Tant sols els immunoreactius de Lp(a) van produir aquestes interferències i per aquest motiu es van descartar.

En aquest treball de recerca va ser impossible quantificar la CRP i Cys C en el mateix microarray que la H-FABP, cTnl i NT-proBNP de mostres directes. Afortunadament, el fet d'utilitzar superfícies de vidre en les quals es podien imprimir fins a 24 microarrays ha permès poder fer aquestes mesures de forma simultània i paral•ela.

Amb el microarray multiplexat ha estat possible mesurar mostres de pacients amb diferents patologies. Malauradament, no va ser possible mesurar els nivells de cTnl amb aquest microarray, tal com era de preveure d'acord amb els estudis previs fets amb els immunoreactius utilitzats. Així doncs, podem considerar aquest microarray com un mètode semi-quantitatiu multiplexat útil per a la millora del diagnòstic de malalties cardiovasculars.

Finalment, s'han realitzat estudis preliminars per implementar el sistema immunoquímic multiplexat en un sensor òptic fluorescent d'ona evanescent amb l'objectiu d'aconseguir un dispositiu POC (point-of-care). Malauradament, els resultats obtingut apunten a que és necessari fer un major esforç per a incrementar la detectabilitat d'aquest sistema, donat que els valors de LOD assolits són pitjors que els aconseguits amb l'ELISA o el microarray i, per casos com l'NT-proBNP, es troben molt allunyats dels valors basals.