Selvarajah, Senthooran
(2013)
The development and application of an antibody microarray as a diagnostic platform for COPD.
PhD thesis, University of Nottingham.
Abstract
According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) Management Guidelines (2001), the definition of COPD is “a disease state characterised by airflow limitation that is not fully reversible. The airflow is usually progressive and associated with inflammatory responses of the lungs to noxious particles and gases.” It is becoming an increasing prevalent problem worldwide, with the incidences of morbidity and mortality continually increasing and promoting a lower quality of life in individuals that continue to suffer from it.
To date, there is still an incomplete understanding of the pathogenesis of the disease resulting in poor diagnosis and treatment plans for COPD that are insufficient in preventing a decline in lung function. In recent years, research has focussed on discovering a set of biomarkers that could improve our understanding of pathogenesis of disease. The ability to measure a vast array of biomarkers simultaneously is highly desirable however the cost associated is somewhat prohibitive. Current methods centre on measuring the presence or absence of multiple biomarkers in patient samples compared to controls.
As COPD is a multi-component disease which encompasses diseases such as emphysema and chronic bronchitis, it may be necessary to look at biomarker patterns within each disease category. A variety of immune effector cells are known to lead to the pathophysiology of COPD including neutrophils, macrophages and CD8 T-lymphocytes that are all documented to be increased in number and contribute to the inflammatory process.
Protein microarrays are used as a measurement tool to determine and quantify the presence or amount of proteins that exist in biological samples (i.e. blood, sputum, [iii] urine etc). The wide use of protein microarray technology has advanced diagnosis and management of multifactorial diseases such as cancer, autoimmunity and allergy.
At present, multiple microarray kits are available to researchers at a large cost which make it impractical for most research groups to investigate multiple biomarkers of interest simultaneously. Here we show development, validation and implementation of our bespoke in-house microarray platform enabling quantitative and simultaneous analysis of multiple protein biomarkers at a reasonable cost. The methodology is based on the traditional sandwich ELISA; antibodies are immobilised on poly-L-lysine coated glass and signals amplified and quantified through fluorescence. The accuracy and reproducibility of the in-house microarray was investigated using the guidelines outlined by the Food and Drug Administration (FDA) for pharmacokinetic assay validation. The assay was shown to have high reproducibility with assay accuracy between 80-120% and precision within 20% coefficient of variation, except in very low abundant cytokines such as IL-10, where the CVs were higher due to the variation at the lowest concentrations in sera. Importantly there were no significant differences between ELISA and microarray.
This microarray platform was then used to study a selection of healthy controls (n=12), healthy smoking controls (n=36) and COPD patients (n=60) to see if there was a difference in the expression of the 16 biomarkers tested. The overall analysis of the 16 biomarkers investigated in this study, a significant increase in expression of eotaxin-2 was observed in the sera those that have COPD compared to healthy controls and healthy smoking controls. This suggests that eoxtaxin-2 may potentially be responsible for the recruitment and activation of multiple cytokines which in turn lead to the inflammatory cascade observed in COPD
COPD severity is divided into four categories according to international guidelines outlined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD). This is often known as stage 1 (mild), stage 2 (moderate), stage 3 (severe) and stage 4 (very severe). This is based on the forced expiratory volume per second (FEV-1%). Interestingly when investigating the different severities of GOLD in COPD, it was observed that at the highest stage of GOLD (stage 4), the expression of 15 of the 16 biomarkers had dropped significantly in comparison to the other stages. This may suggest that at this point of the disease process, the immune system may in fact be suppressed in alliance to hypoxia experienced by an individual.
Additionally it has to be acknowledged that the medication that the COPD patients were on were not available prior to analysis. It has to be taken into account that patients at GOLD stages 3 and 4 could be likely to be on a high dose of inhaled corticosteroids, which are immunosuppressive which would lead to drop in the 15 cytokines observed. However without the information available, it cannot be definitive to make such conclusions.
Hence this work offers an understanding into the development of a bespoke microarray platform that is capable of investigating protein biomarkers in any disease setting.
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