Principle of Method
This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human FGL1 in cell culture supernatants, serum and plasma. A monoclonal antibody specific for human FGL1 has been precoated onto the 96-well microtiter plate. Standards (STD) and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, FGL1 (h) is recognized by the addition of a biotinylated monoclonal antibody specific for human FGL1 (DET). After removal of excess biotinylated antibody, streptavidin-peroxidase (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 FGL1 in the samples.
FGL-1 (Fibrinogen-like protein 1, also called Hepatocyte-derived fibrinogen-related protein 1, HFREP-1 or Hepassocin) was initially identified as an overexpressed transcript in hepatocyte carcinoma (1) and as a transcript enriched in regenerating liver (2). Under physiological conditions, FGL-1 is a liver secreted factor and contributes to its mitogenic and metabolic functions (3), but it has also been shown to be expressed at lower levels in brown adipose tissue in the setting of liver injury (4). Structurally, FGL-1 is a 34 kDa protein similar to Angiopoietin-like factors 2, 3, 4 and 6, regulating also lipid metabolism and energy utilization. Thus, it was proposed to be a member of an emerging group of proteins with key roles in metabolism and liver regeneration (4).
Recently, FGL-1 has also been shown to be upregulated in human cancers, especially non-small cell lung carcinomas (NSCLC), acting as a functional inhibitory ligand of lymphocyte-activation gene 3 (LAG-3) (5). FGL-1/LAG-3 interaction occurs in a MHC-II-independent manner and involves the FGL-1 fibrinogen-like domain (FD) and the LAG-3 D1-D2 domains (5). FGL-1 forms two disulfide-linked homodimers (2) and higher molecular weight oligomers that bind to LAG-3 much better than dimeric forms (5). Thus, it acts as an immune suppressive molecule inhibiting antigen-specific T-cell activation and being responsible for LAG-3 T-cell inhibitory function (5). Because FGL-1/LAG-3 interaction is independent from the B7-H1-PD-1 pathway, elevated FGL-1 in the plasma of cancer patients is associated with poor prognosis and resistance to anti-PD therapy (5).
Sandwich type ELISA
HP-041, Hepassocin (HPS), Hepatocyte-derived fibrinogen-related protein 1 (HFREP-1), Liver fibrinogen-related protein 1 (LFIRE-1)
Serum, Plasma or Cell Culture Supernatant
This ELISA is specific for the measurement of natural and recombinant human FGL1.
Serum and Plasma : 0.1 µL (actual amount of sample loaded in well after dilution)
Calibration Range: 7.78 pg/ml – 500 pg/ml
Limit of Detection
Intra-assay (Within-Run) C.V.
Inter-assay (Run-to-Run) C.V.
References to Summary
- Molecular cloning and initial characterization of a novel fibrinogen-related gene, HFREP-1: T. Yamamoto, et al.; BBRC 193, 681 (1993)
- Isolation and characterization of a novel liver-specific gene, hepassocin, upregulated during liver regeneration: H. Hara, et al.; Biochim. Biophys. Acta 1492, 31 (2000)
- Recombinant human hepassocin stimulates proliferation of hepatocytes in vivo and improves survival in rats with fulminant hepatic failure: C. Li, et al.; Gut 59, 817 (2010)
- Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization: V. Demchev, et al.; PloS One 8, e58084 (2013)
- Fibrinogen-like Protein 1 Is a Major Immune Inhibitory Ligand of LAG-3: J. Wang, et al.; Cell 176, 334 (2019)