Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used for determination of serum biomarker levels. In many instances, following a pilot study, serum samples had to be diluted (10–50 fold in saline) and the ELISAs rerun so that sample levels remained within the standard curve. The HCC biomarkers evaluated (catalog number, source of ELISA kit) are as follows: Alpha fetoprotein (AFP) (MAFP00, R&D), Lens culinaris A-reactive fraction of AFP (AFP-L3) (MBS724605, MYBioSource), Des γ carboxyprothrombin (DCP / PIVKA-II, MBS2516006; MYBioSource), Glypican-3 (MBS705612, MYBioSource), Osteopontin (OPN, MOST00; R&D), Golgi protein-73 (GP73, MBS024709; MYBioSource), Dickkopf-related protein 1 (DKK1, DY1765; R&D) Serum cystatin C was measured using an ELISA kit (MSCTC0, R&D). Serum aspartate transaminase (AST) and serum alanine transaminase (ALT) levels were measured by a commercial laboratory (Northwell Health, NY).
Mafp00
Manufactured by R&D Systems
Sourced in United States
MAFP00 is a laboratory equipment product. It is designed for use in research and development settings. The core function of MAFP00 is to [DESCRIPTION NOT AVAILABLE].
Automatically generated - may contain errors
5 protocols using mafp00
1
Serum Biomarker Profiling for HCC
2
Serum Biomarker Quantification in Mice
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Serum was isolated using Microvette 500 Z-GEL Gel/clotting activator tubes (Sarstedt AG & Co. KG, Nümbrecht, Germany) per manufacturer’s instructions. Levels of α-fetoprotein (AFP), alanine aminotransferase (ALT), and bilirubin were detected in serum of 32- and 48-week-old mice using commercially available kits (AFP, catalog number MAFP00, R&D Systems, Inc., Minneapolis, Minnesota, USA; ALT, catalog number ab282882, Abcam, Berlin, Germany; bilirubin, catalog number NBP2-69939, Bio-Techne GmbH, Wiesbaden Nordenstadt, Germany) per manufacturer’s protocols.
3
Quantification of Serum AFP and Cytokines
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AFP was quantified by Enzyme Linked Immunosorbant Assay (ELISA, R&D Systems, MAFP00) on serum according to manufacturer’s instructions. Cytokines were quantified by ELISA (Invitrogen ref 88-7314-22 for IFN-γ and Invitrogen 88-7064-22 for IL6) on culture supernatants from peritoneal macrophages according to manufacturer's instructions.
4
Measuring Serum Biomarkers in Mice
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Mouse-specific ELISA kits were used to measure circulating alpha-fetoprotein (AFP) (MAFP00; R&D Systems, St. Louis, MO), IGF-I (MG100; R&D Systems), and GH (EKU04609; Bio-Matik, Wilmington, DE). Briefly, serum samples were diluted according to the manufacturer’s recommended protocols. Standards and samples were used simultaneously in each experimental setup. Optical densities were measured using a microplate reader (CLARIOstar; BMG Labtech, NC). Serum concentrations were calculated according to equations of linear standard curves generated by plotting optical densities and standard concentrations.
5
Plasma Biomarkers for Liver Health
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Non-fasted terminal blood was sampled from the tail vein, kept on ice and centrifuged (5 min, 4 °C, 6000× g) to generate EDTA-stabilized plasma. Plasma lipids (triglycerides, total cholesterol) as well as markers of liver injury (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and caspase-cleaved cytokeratin CK18 (CK18-M30) were determined as described previously18 (link),97 (link). Plasma markers of fibrosis included amino-terminal propeptide of type III procollagen (PIIINP, #CSB-E08095m, CusabBio, Houston, TX) and tissue inhibitor of metalloproteinase-1 (TIMP-1). PIIINP was determined according to the manufacturer’s instructions, TIMP-1 was assayed as described previously18 (link). Alpha-fetoprotein was determined according to the manufacturer’s instructions (#MAFP00, R&D Systems, Minneapolis, MN).
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