Halala, Ramadan
(2017)
Profiling natural autoantibodies in relation to chronic obstructive pulmonary disease.
PhD thesis, University of Nottingham.
Abstract
Chronic obstructive pulmonary disease (COPD) is a complex condition involving chronic inflammation of the airways (chronic bronchitis) and destruction of airways parenchyma (emphysema). It is characterized by progressive airway limitation that is not fully reversible. Although the precise mechanisms of disease remains unclear, it has been thought that noxious materials, such as gases caused by smoking, may play role in the pathogenesis of the disease. However, only 15-20% of smokers developed COPD and non-smokers have also developed the disease, suggesting that other determinants may be involved the disease onset and other contributing factors may be exist. One such factor may be autoimmunity. COPD is accepted as an inflammatory disorder with evidence of circulating autoantibodies against variety of targets. Natural autoantibodies (nAAbs) are autoantibodies found in the circulation of healthy individuals without development of autoimmunity and they are considered to play a role in immune regulation and act as a protector in healthy individuals. However it has been suggested that nAAbs, under circumstances, may shift to be pathological autoantibodies.
The aim of the study was to investigate nAAbs in healthy individuals in relation to COPD. To achieve the project aim; firstly ELISA was used to detect reactivity of IgG , IgM and IgA nAAbs in 23 healthy non-smoker individuals to four auto antigens. However, ELISA technique has some limitations. It requires high sample volume and takes considerable time. In order to perform our goal, that is to investigate 37 auto antigens and screen numerous healthy individuals and COPD patients’ samples in the limited time of this PhD project, a high throughput, multiplexed assay was required.
Therefore, we moved to develop an antigen microarray platform that would be a high throughput technique that is rapid, accurate, automated and highly sensitive. Before applying this technique, quality control experiments were performed to optimize the procedures, including testing different slides surface coatings, investigating numerous blocking buffers, as well as series of validatory tests to determine if the assay was both accurate and reproducible.
The optimization results showed that a combination of aminosilane coated slides and I-block buffer performed well and gave high signal intensity and low backgrounds. Validation tests were performed for the optimized technique and showed no cross reactivity occurred, coefficient of variation (CV%) was acceptable, and the limit of detection (LoD) was satisfactory.
This validated technique was used to then screen natural autoantibody responses to a range of autoantigens in healthy non-smokers and COPD samples. The study compared purified and unpurified IgG Abs in healthy individuals and COPD patient samples and showed significance differences in reactivity of IgG Abs in most auto antigens investigated in healthy subjects. Conversely, no significance differences were detected in autoantibody responses to most auto antigens investigated in COPD patients samples suggesting serum components may be involved in controlling the auto reactivity of natural IgG Abs in healthy individuals.
Natural IgM Abs was also investigated against 37 auto antigens in healthy individuals and COPD patient samples and the results showed various pattern of reactivity of IgM Abs to all auto antigens suggesting that natural IgM Abs may play a role in controlling the auto reactivity of natural IgG Abs in healthy individuals. This control may be achieved via either anti-idiotypic activity of the natural IgM Abs or could be in part dependent on the competitive binding of IgM Abs to the auto antigens.
Regarding comparison of autoantibody responses to 37 auto antigens in COPD patients compared to healthy individuals, the results showed there was a significance increased in reactivity for both IgG and IgM auto antibodies in COPD group compared to healthy subjects for Elastin, ATS 0.02/sm, HNRPD, HNRP2B1, Keratin8, PL-12, Vimentin, BPI, Vitronectin, Collagen V and Lactoferrin. This suggests that these auto antigens have the potential to be used as a diagnostic biomarker for the detection of COPD or tracking disease progression.
In conclusion, nAAbs showed various pattern of reactivity to all auto antigens investigated in this study with high levels observed in reactivity of purified IgG Abs in healthy individuals. This suggests that a serum component may be involved in regulation of auto reactivity of natural IgG Abs in normal conditions. This thesis has also developed a rapid, inexpensive, broad-spectrum antigen microarray technology, which may have an important future role in monitoring the levels of nAAbs and early diagnosis of COPD.
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