Principle of Method
This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of mouse or rat ANGPTL3 in biological fluids. A polyclonal antibody specific for ANGPTL3 has been precoated onto the 96-well microtiter plate. Standards and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, ANGPTL3 is recognized by the addition of a biotinylated polyclonal antibody specific for ANGPTL3 (Detection Antibody). After removal of excess biotinylated antibody, HRP labeled streptavidin (STREP-HRP) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of ANGPTL3 in the samples.
The angiopoietins are a family of growth factors that are specific for vascular endothelium. Conklin et al. (1) isolated a full-length cDNA encoding angiopoietin-like protein 3 (ANGPTL3) from a human fetal liver/spleen cDNA library. The deduced 460-amino acid ANGPTL3 protein has the characteristic structure of angiopoietins: a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils, a short linker peptide, and a globular fibrinogen-like domain (FLD). Human ANGPTL3 shares 76% amino acid sequence identity with mouse Angptl3. Northern blot analysis of human tissues showed a preferential expression of 4 ANGPTL3 transcripts being 4.5, 3.0, 2.8, and 1.7 kb in liver.
Camenisch et al. (2) showed that ANGPTL3 induced angiogenesis in a rat corneal assay. ANGPTL3 is structurally similar to angiopoietins, which are vascular endothelial growth factors. The experimental results show that Angptl3 and Angptl4 function to regulate circulating triglyceride levels during different nutritional states and therefore play a role in lipid metabolism during feeding/fasting through differential inhibition of Lipoprotein lipase (LPL). Overexpression of Angptl3 or intravenous injection of the purified protein in KK/San mice elicited an increase in circulating plasma lipid levels. These data suggested that Angptl3 regulates lipid metabolism in animals. The authors suggested the possibility that genetic variation in ANGPTL3 contributes to atherosclerosis, coronary artery disease, and diabetes mellitus. In vitro analysis of recombinant protein revealed that Angptl3 directly inhibits both endothelial lipase and lipoprotein lipase (LPL) activity (5, 6). Another line of evidence suggests that ANGPTL3 play an important role in regulation of HDL synthesis (7). The implication of ANGPTL3 in a number of metabolic dysfunctions suggests that ANGPTL3 is a novel predictor of these. Given these findings, ANGPTL3 has potential as a novel therapeutic target for treatment of hyperlipidemia.
Sandwich type ELISA
Angiopoietin-like protein 3,
Serum, Plasma or Cell Culture Supernatant
This ELISA is specific for the measurement of natural and recombinant mouse or rat ANGPTL3. It does not cross-react mouse ANGPTL4, mouse adiponectin, mouse resistin, mouse RBP4, mouse vaspin, mouse Nampt, mouse clusterin, mouse GPX3, mouse progranulin, mouse IL-33, mouse leptin, mouse RELM-β, human ANGPTL3, human ANGPTL4, human Nampt, rat Nampt.
Mouse Serum and Plasma : 0.025 µL (actual amount of sample loaded in well after dilution)
Rat Serum and Plasma : 0.25 µL (actual amount of sample loaded in well after dilution)
Calibration Range: 0.156 ng/ml – 1 ng/ml
Limit of Detection
Intra-assay (Within-Run) C.V.
Inter-assay (Run-to-Run) C.V.
References to Summary
- Identification of a mammalian angiopoietin-related protein expressed specifically in liver: D. Conklin, et al.; Genomics 62, 477 (1999)
- ANGPTL3 stimulates endothelial cell adhesion and migration via integrin alpha-v-beta-3 and induces blood vessel formation in vivo: G. Camenisch, et al.; J. Biol. 277, 17281 (2002)
- Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells: C.C. Zhang, et al.; Nature Med. 12, 240 (2006)
- Angptl3 regulates lipid metabolism in mice: R. Koishi, et al.; Nature Genet. 30, 151 (2002)
- ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase: T. Shimizugawa, et al.; J. Biol. 277, 33742 (2002)
- Angiopoietin-like protein3 regulates plasma HDL cholesterol through suppression of endothelial lipase: M. Shimamura, et al.; Arterioscler. Vasc. Biol. 27, 366 (2007)
- Hepatic proprotein convertases modulate HDL metabolism: W. Jin, et al.; Cell Metab. 6, 129 (2007)