Profile identification of disease-associated humoral antigens using AMIDA, a novel proteomics-based technology

Abstract

We describe AMIDA (autoantibody-mediated identification of antigens), a novel target identification technology based on the immunoprecipitation of disease-specific antigens by autologous serum antibodies followed by two-dimensional electrophoretic separation, and their identification via mass spectrometry. Twenty-seven potential carcinoma antigens were identified including proteins of hitherto unknown function. Validation of one of the identified antigens, cytokeratin 8, revealed its de novo expression in hyperplastic tissue, gradual overexpression with increasing malignancy, and ectopic localization on the cell surface. Furthermore, a strong prevalence of CK8-specific antibodies occurred in the serum of cancer patients already at early disease stages. In situ hybridization for one marker of unknown function, KIAA1273/TOB3, demonstrated its strong overexpression in head and neck carcinomas, thus making it a likely tumor antigen candidate. Eventually, AMIDA could foster significant improvements for the diagnosis and therapy of human diseases eliciting a humoral immune response, and allows for the rapid identification of new target molecules.

Human hair follicle and interfollicular keratinocyte reactivity to mouse HPV16-transformed cells: An in vitro study

“…Under pathological conditions this has been observed in some basal cell carcinomas (40). Keratin 8 which is induced by TC-1 is not expressed by the cells of squamous epithelia in the postnatal period under physiological conditions but its expression in the squamous cell carcinoma has been recognized as a marker of poor clinical prognosis of patients (44). The two keratins are not expressed in human keratinocytes during the prolonged cultivation in vitro except for the keratinocytes cocultured with stromal cells from basal or squamous cell carcinomas exhibiting the presence of keratins 8 or 19 (22,23).…”

Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases

“…this approach, which is called autoantibody mediated identification of antigens (AMIDA) (35), patient/control immunoglobulins are first bound to Protein-A/Protein-G magnetic beads; relatively large amounts of whole cell/ tissue protein lysates containing potential autoantigens are then co-incubated with the antibody-coated beads. Unbound proteins are washed away, allowing the bound, putative autoantigen proteins to be eluted.…”

“…The vertical axis represents the integrated protein abundance in ppm in a logarithmic scale. PPM (parts per million) is the unit of abundance that is used to quantify relative abundance within the proteome (35). (A) The horizontal axis represents each protein discovered using SERPA from 24 studies (8,.…”

“…(B) The horizontal axis represents each protein discovered using immuno-affinity capture technologies such as AMIDA, CIC and SELDI-TOF from 12 studies (38-42, 46-52). The vertical axis represents the integrated protein abundance in ppm (35) in a logarithmic scale.…”

Identification of Three New Autoantibodies Associated with Systemic Lupus Erythematosus Using Two Proteomic Approaches

“…The IgG bound proteins were then eluted with 0.1 M glycine-HCl, pH 2.4, and neutralized with 2 M Tris-HCl, pH 8.0, 1.5 mM NaCl, and 1 mM EDTA. The autoantigen proteins thus obtained were desalted and concentrated by methanol/chloroform precipitation (25)(26)(27).…”