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New bioactive angiopoietin-2 ELISA allows the quantification of all three isoforms of angiopoietin-2 in chronic kidney disease
Ana-Maria Suciu Andreea, Jacqueline Wallwitz, Berg Gabriela, Maria Laber Anna, Himmler Gottfried. Nephrology Dialysis Transplantation, Volume 34, Issue Supplement_1, June 2019, gfz103.SP339, https://doi.org/10.1093/ndt/gfz103.SP339
Click here to view poster: ERA-EDTA Poster 2019 Human Angiopoietin-2
INTRODUCTION: Angiopoietin-2 (Ang-2) is an important regulator of the angiopoietin-1/Tie-2 receptor signaling system on endothelial cells during angiogenesis. Disruption of this signaling leads to the loss of endothelial integrity and renders the endothelium response towards a variety of pro-inﬂammatory cytokines and growth factors. Thus, Ang-2 might lead to vascular micro-inflammation in patients with CKD (chronic kidney disease). Ang-2 levels increase with CKD stage, are associated with fluid overload and abnormal cardiac structure and predict mortality in patients with CKD stages 4–5. Although Ang-2 levels return toward normal after successful kidney transplantation, Ang-2 remains a putative cardiovascular risk factor in this population.
METHODS: An enzyme-linked immunosorbent assay for the detection of all three angiopoietin-2 isoforms in human serum and plasma was developed. Two high quality antibodies are combined in a sandwich test format: As capturing antibody a recombinant monoclonal antibody is used. A biotin-labeled polyclonal affinity-purified antibody serves for detection of the analyte. High resolution epitope mapping of the antibodies via peptide microarray technology allowed the identification of linear antibody epitopes. Technical performance and accuracy of the assay were assessed according to ICH/EMEA guidelines.
RESULTS: Microarray data illustrate the binding of the polyclonal detection antibody to human angiopoietin-2 spotted on a chip. Altogether, seven linear epitopes located N-terminal/at the center of angiopoietin-2 were detected. Most relevant linear epitopes are epitope e2 in the super clustering region and e6 near the fibrinogen-like domain, displaying a twofold higher fluorescent signal than the remaining epitopes. For the recombinant monoclonal antibody no fluorescence was recorded. This antibody recognizes a structural epitope within the C-terminus of angiopoietin-2, which covers the bioactive receptor-binding site of the protein. We expect to detect all described angiopoietin-2 isoforms, as the receptor-binding site is conserved and the majority of the polyclonal antibody epitopes are present in all isoforms. This assay is in conformance with ICH/EMEA/FDA guidelines. Validation data demonstrate its applicability in nephrological disorders including chronic kidney disease. Here we compare an apparently healthy population to chronic kidney disease samples on haemodialysis and kidney transplant samples.
CONCLUSIONS: This new angiopoietin-2 ELISA enables a quick and accurate quantification of all human angiopoietin-2 isoforms that are bioactive in chronic kidney disease and other nephrological disorders.
- Molnar et al. (2014): Circulating angiopoietin-2 levels predict mortality in kidney transplant recipients: a 4-year prospective case–cohort study. Transplant International, 27 (6): 541-52. 2. David et al. (2010): Circulating angiopoietin-2 levels increase with progress of chronic kidney disease. Nephrol. Dial. Transplant, 25: 2571-2579,
- Tsai et al. (2017): The interaction between fluid status and angiopoietin-2 in adverse renal outcomes of chronic kidney disease. PLoS One, 12 (3): e173906.
- Tsai et al. (2016): Angiopoietin-2, Angiopoietin-1 and subclinical cardiovascular disease in Chronic Kidney Disease. Scientific Reports, 6:39400