Dialyzed sample was concentrated using an Amicon Ultra centrifugal concentrator (Millipore, USA) with 8C14 kDa molecular weight cutoff. (IPTG) and the purified sFGL1 was used as an antigen to produce mouse monoclonal antibody (mAb) and rabbit polyclonal antibody (pAb). After identification, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for sensitive and specific detection of sFGL1 was developed. Swine FGL1 in samples was captured by anti\sFGL1 mAb followed by detection with anti\sFGL1 rabbit pAb and HRP-conjugated goat anti-rabbit IgG. The limit of detection of the developed sFLG1-DAS-ELISA is usually 35 pg/ml with recombinant sFLG1. Besides, it does not Kinesore show cross\reactivity with the control protein. Then serum samples of PRRSV-negative and -positive pigs were tested with the established DAS-ELISA and calculated according to the equation of y=0.0735x+0.0737. The results showed that PRRSV contamination enhanced the serum FGL1 levels significantly. Our research provides a platform for the research around the functional functions of swine FGL1. Keywords: swine FGL1, detection, monoclonal antibody, double-antibody sandwich ELISA, PRRSV-negative and -positive pig sera Highlights Fibrinogen-like protein 1 (FGL1) is usually a major ligand of lymphocyte activating gene 3 (LAG3) and the FGL1CLAG3 conversation reveals a new immune escape mechanism. Our double-antibody sandwich ELISA allows sensitive and Prkwnk1 specific detection of swine FGL1 in serum samples which can provide technical support for exploring the role of FGL1 in immunosuppressive diseases of pigs. Introduction Fibrinogen-like protein 1 (FGL1), also known as hepatocyte-derived fibrinogen-related protein 1 Kinesore (HFREP1) or Hepassocin (HPS), is usually a hepatocyte secreted protein that was initially cloned from liver tissue and has been demonstrated to be over-expressed in human hepatocellular carcinoma (1C3). This protein belongs to the fibrinogen superfamily and it contains a fibrinogen-related domain name in its C-terminal portion but lacks three functional domains of platelet binding site, crosslinking region, and thrombin-sensitive site (4, 5). The exact role of FGL1 in the liver is controversial. It has been reported that exogenous FGL1 promotes the proliferation of normal hepatocytes, stimulates hepatocyte proliferation and (for 10?min at 4C. After that, the cell pellet obtained from 1 L culture was resuspended in 25?ml PBS and sonicated on ice. After centrifugation at 15,000for 20?min at 4C, the precipitate was resuspended with solubilization buffer(100 mmol/L Na2PO4, 10 mmol/L TrisCHCl, 8 mol/L urea, pH 8.0) and the targeted protein was purified with cOmplete? His-Tag Purification Resin according to the instructions (Roche, Switzerland). The expression and the purification effect were analyzed by sodium dodecyl sulfate-polyacrylamide Kinesore gel electrophoresis (SDS-PAGE) and Western blot. Eluates made up of recombinant sFGL1 were pooled and dialyzed. Dialyzed sample was concentrated using an Amicon Ultra centrifugal concentrator (Millipore, USA) with 8C14 kDa molecular excess weight cutoff. The protein concentration of the recombinant sFGL1 was decided with a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Open in a separate window Plan 1 Graphic abstract. (A) Diagram for the acquisition of the mouse mAb and rabbit pAb against sFGL1. (B) Designation of the developed DAS-ELISA. Table 1 Kinesore Primers used in this study. FGL1 sequence on NCBI (NCBI reference sequence: XM_021077953.1), and the derived amino acid sequence was completely consistent Kinesore with the logged FGL1 (NCBI reference sequence: XP_020933612.1). A, G, T, and C were 301, 237, 242, and 171, respectively, with A + T accounting for 57.10% and G + C for 42.90%. The nucleotide sequence was submitted to the GenBank database of NCBI, and the GenBank sequence number is usually MK813968. The sFGL1 gene without signal peptide sequences is usually 873 bp ( Physique 1B ). This fragment was ligated in pQE-30 vector and the recognized plasmid pQE30-sFGL1 was transformed into JM109 qualified cells. After induced with 1 mmol/L IPTG at 37 C for 6?h, recombinant sFGL1 protein was about 35 kDa and mainly expressed in the form of inclusion bodies ( Physique 1C ). When detected by Western blot with (expression system. After identification and purification, pAbs were prepared by immunizing New Zealand rabbits and mAbs were prepared by immunizing BALB/c mice and cell fusion technique. Finally, pAbs with titers of 1 1:102,400 ( Physique 2 ) and two mAbs, 2D7 and 4G7 ( Physique 3 ), were obtained. In Western blot identification of recombinant sFLG1, there were also poor imprinted bands in the lane of the no-induced.