Autoantibodies of IgM and IgG classes show differences in recognition of multiple autoantigens in chronic obstructive pulmonary disease

Autoimmunity occurs in chronic obstructive pulmonary disease (COPD). We describe an antigen microarray for detecting serum autoantibodies (AAbs) to determine how IgM, as well as IgG, AAbs distinguish patients with COPD from controls with a history of smoking without COPD. All COPD patients' sera contained elevated levels of AAbs to some of 30 autoantigens. There were significant differences in the autoantigenic specificities of IgM AAbs compared to IgG AAbs in the COPD sera: for example, AAbs to histone and scl-70 were mainly IgG, whereas AAbs to CENP-B and La/ssB were mainly IgM; by contrast, IgM and IgG AAbs to collagen-V were equally prevalent. Thus, a combination of IgM and IgG AAbs specific for multiple autoantigens are detected in all cases of COPD at a level at which all non-COPD controls are negative for AAbs. This highlights the importance of different classes of AAbs to a range of autoantigens in COPD

Profiling autoantibodies in chronic obstructive pulmonary disease using antigen microarray technology

Chronic obstructive pulmonary disease (COPD) encompasses the diseases of chronic bronchitis and emphysema, both of which can cause the main feature of this disease; airflow obstruction. COPD is the third leading cause of mortality in the world, and although cigarette smoking is the main risk factor of the disease, other environmental factors and genetic disorders can also play a role. The pathogenesis of COPD is currently still poorly documented, which results in poor diagnosis and treatment for the disease. The involvement of autoimmunity in the pathogenesis of the COPD is becoming more apparent; in recent studies, high levels of circulating autoantibodies have been detected in patients with COPD, suggesting that this lung disorder may have an autoimmune component. Therefore, research has focused on identifying a set of autoantigens known to be associated with other autoimmune disorders that may play a role in the pathogenesis of this disease. The aim of this study was to develop antigen microarrays for profiling antigen specific-autoantibodies in the serum of patients with COPD, as a diagnostic tool at the early stage of the disease. To achieve the project aim, we have employed antigen microarrays. Before applying this technique, quality control experiments were performed to optimize the procedure. This included testing different slide surface coatings, investigating numerous blocking buffers and methods to amplify the signal intensities, as well as a series of validatory tests to determine if the assay was both accurate and reproducible. The optimization results showed that a combination of amino-silaine coated slides with I Block buffer and Genisphere amplifier performed well due to the high signal detection and low background observed. Validation for optimized technique was achieved and showed no cross-reactivity occurred, an acceptable limit of the coefficient of variation (CV %), and a significant correlation between ELISA and the antigen microarray platform, with microarray giving a bigger dynamic range of signals. The results for the limit of detection (LOD) were applied and the cut off for a positive autoantibody response was taken from the 95th percentile of the healthy non-smokers control group. The results for the investigation of the different 39 autoantigens showed that there was a significant increase in reactivity for both the IgG and IgM autoantibodies in the COPD group compared to the control for CENP-B, collagen5, RNP/sm, La ssb, histone, ro-52 and SCL-70, which suggests these antigens have the potential to be used as diagnostic biomarkers for the detection of COPD. These results also showed that a number of healthy smokers produced an increased reactivity to the same autoantigens as the COPD patients, which suggests there is potential susceptibly of these smokers to develop COPD. In conclusion, this thesis developed a rapid, inexpensive, broad-spectrum antigen microarray technology, which could have a pivotal future role in the early diagnosis of COPD