AbstractsBiology & Animal Science

Development and Diagnostic Application of a Novel Quantitative Test for Determination and Characterization of Autoantibodies against Insulin-like Growth Factor 1 Receptor

by Tim Welsink

Institution: Freie Universität Berlin
Department: FB Biologie, Chemie, Pharmazie
Degree: PhD
Year: 2015
Record ID: 1105691
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000099099


Autoantibodies (aAb) are characteristic for autoimmune diseases. Specific aAb are often found both in diseased and apparently healthy individuals and may precede pathological symptoms. The contribution of thyrotropin receptor (TSHR) stimulating aAb to the progression of Graves´ Orbitopathy (GO) is widely accepted. The coexistence of stimulating insulin-like growth factor 1 receptor (IGF1R) aAb in GO patients was hypothesized and tested in the present study. As no method for routine testing was available, a suitable non-radioactive test was established to evaluate IGF1R-aAb in human sera and test the hypothesis. As a first step, an IGF1R-aAb precipitation assay for detection and reliable quantification of these aAb was developed. This work package included the genetic engineering and expression of the recombinant antigen, its purification and characterization, the definition of a standardized operating procedure, and the adaptation of the assay format for future routine use. Assay development also includes the generation and purification of monoclonal antibodies (mAb) to the IGF1R, needed for standardization and supplementary characterization of the assay. A central part of this work focuses on the clinical, biological and molecular role of IGF1R aAb. To this end, aAb were purified from human sera and characterized in vitro. The IGF1R-aAb inhibit signal transduction via the IGF1R directly and acted as antagonists of IGF1-induced proliferation of MCF7 breast cancer cells in culture An analysis of IGF1R protein domains indicates that IGF1R aAb in human sera are polyclonal and often recognize conformational epitopes. As IGF1R aAb were detected with a prevalence of approx. 10% in human subjects, and immunoglobulins are known to contain two antigen binding sites, a second more direct and convenient assay type was developed for routine diagnostic use. A cohort of GO patients were analyzed yielding a prevalence of 10% positive IGF1R-aAb carriers, but not different from the prevalence in healthy controls. Moreover, IGF1R-aAb were not stimulating the IGF1R. Hence, the initial hypothesis that IGF1R-Ab contribute to GO pathogenesis needs to be rejected as it was not supported by the data. Nevertheless, this study demonstrates a high prevalence of aAb against the IGF1R in pathological samples and apparently healthy subjects as well, which might be of clinical importance given the central role of IGF1 in growth, metabolism and disease. In cooperation with a fellow PhD student who established an analogous assay for the structurally-related insulin receptor (IR), an analysis of the specificity of the IGF1R-aAb was performed. We realized that roughly 50% of IGF1R-aAb positive samples cross-reacted with the IR, highlighting their potential clinical importance for a specific form of diabetes mellitus, i.e., autoimmune type B insulin resistance. Further studies with samples from well characterized samples, respective pre- and diabetic patients, and controls are needed to clarify this notion. Collectively, two novel analytical…