Abstract
Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases (Wang et al., 2015). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed an ELISA-based quantitative assay to detect the presence of autoantibodies as well as to measure the circulating autoantibodies in the sera of patients suffering from large vessel vasculitis (LVV), an autoimmune disease (Chakravarti et al., 2015). Using this assay, we detected the increase in anti-aortic antibodies in LVV patient’s sera. We have further verified the results by independent biochemical techniques and found the specificity to be > 94% (Chakravarti et al., 2015). This method can be uniquely modified to suit any autoimmune, in particular organ specific, disease and thus has wider applications in the detection and quantification of autoantibodies.
Keywords: ELISA, Autoantigen, Autoantibody, Vasculitis, Serum, Autoimmune disease
Background
There are about 80-100 autoimmune diseases wherein immune cells recognize a self-protein as a foreign antigen, and get activated to generate humoral response. Though the trigger for most autoimmune diseases remains a mystery, presence of higher levels of antibodies in the patient’s sera is very common (Wang et al., 2015). Some of the examples include antibodies against neutrophil cytoplasmic antigens in small vessel vasculitis, myelin basic protein in multiple sclerosis, glutamate decarboxylase in type 1 diabetes etc. (Wang et al., 2015). Limited knowledge of autoantigen in most autoimmune diseases presents a challenge for both the detection of disease as well as understanding the pathogenesis. However, ability to detect autoantibodies in the sera provides a diagnostic and prognostic advantage. Discovering a reliable autoantigen in autoimmune diseases are needed to make better clinical decisions. LVV is a spectrum of autoimmune diseases affecting aorta or its primary branch vessels. Like many others, its etiology and cure are not known (Buttgereit et al., 2016). We have developed a qualitative and quantitative assay to detect the presence of autoantibody in the human sera targeting against the human aortic antigen. Our assay provides a tool to find autoantigen in any affected/damaged tissue and in any disease model. We looked for the presence of autoantigen in the human thoracic aortic aneurysms using patients’ sera and tested the specificity of the assay by comparing the sera obtained from related subsets of autoimmune or non-immune diseases. We utilized discarded aortic tissues from aortic reconstruction surgeries and made soluble extracts of aorta to set up the assay (Figure 1). Using this method we performed a high throughput screen for detecting autoantibody in more than 100 sera against aortic antigen (Chakravarti et al., 2015).Figure 1. Schematic Flow Diagram of ELISA to detect autoantibody in human tissue lysates. Step-by-step outline of the method used to detect and quantify autoantibodies present in the sera against antigen present in tissue.
Materials and Reagents
Equipment
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Procedure
Data analysis
Notes
Recipes
Acknowledgments
We are thankful to American Heart Association Scientist Development Grant (15SDG2308025) and the University of Toledo start-up funds to RC. The protocol and result are adopted from our previously published work (Chakravarti et al., 2015) which was carried out at Cleveland Clinic.
References
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