A Double-Antibody Sandwich ELISA for Sensitive and Specific Detection of Swine Fibrinogen-Like Protein 1.

Xin Zhang,Lulu Ning,Haipeng Zhu,Yang Mu,Yunjie Jiao,Xu Zheng,En-Min Zhou

doi:10.3389/fimmu.2021.670626

Abstract

Fibrinogen-like protein 1 (FGL1), a member of the fibrinogen family, is a specific hepatocyte mitogen. Recently, it has been reported that FGL1 is the main inhibitory ligand of lymphocyte activating gene 3 (LAG3). Furthermore, the FGL1-LAG3 pathway has a synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway and is regarded as a promising immunotherapeutic target. However, swine FGL1 (sFGL1) has not been characterized and its detection method is lacking. In the study, the sFGL1 gene was amplified from the liver tissue of swine and then inserted into a prokaryotic expression vector, pQE-30. The recombinant plasmid pQE30-sFGL1 was transformed into JM109 competent cells. The recombinant sFGL1 was induced expression by isopropyl-β-d-thiogalactoside (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 35 pg/ml with recombinant sFLG1. Besides, it does not 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 infection enhanced the serum FGL1 levels significantly. Our research provides a platform for the research on the functional roles of swine FGL1.

Highlights

Fibrinogen-like protein 1 (FGL1), known as hepatocyte-derived fibrinogen-related protein 1 (HFREP1) or Hepassocin (HPS), is a hepatocyte secreted protein that was initially cloned from liver tissue and has been demonstrated to be over-expressed in human hepatocellular carcinoma [1,2,3]
Several well-characterized assays to detect FGL1 are available for humans and mice samples; sFGL1 has not been well characterized
The sFGL1 gene without signal peptide sequences is 873 bp (Figure 1B). This fragment was ligated in pQE-30 vector and the identified plasmid pQE30-sFGL1 was transformed into E. coli JM109 competent cells

Summary

Introduction

Fibrinogen-like protein 1 (FGL1), known as hepatocyte-derived fibrinogen-related protein 1 (HFREP1) or Hepassocin (HPS), is a hepatocyte secreted protein that was initially cloned from liver tissue and has been demonstrated to be over-expressed in human hepatocellular carcinoma [1,2,3]. FGL1 is present in the plasma of rats and a stable fraction of it remains free in the serum at all times This unbound fraction may have other biologic roles distinct from that in liver regeneration and clot formation [2]. FGL1 might be a novel therapeutic target to combat obesity [12]

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Results

Conclusion