A Double-Antibody Sandwich ELISA for Sensitive and Specific Detection of Swine Fibrinogen-Like Protein 1.
A Double-Antibody Sandwich ELISA for Sensitive and Specific Detection of Swine Fibrinogen-Like Protein 1.
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DOI：
10.3389/fimmu.2021.670626
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发表时间：
2021
影响因子：
7.3
通讯作者：
Mu Y
中科院分区：
文献类型：
--
作者：
Zhang X;Zhu H;Zheng X;Jiao Y;Ning L;Zhou EM;Mu Y
纤维蛋白原样蛋白1 （FGL1）是一种特殊的肝细胞丝裂原，是纤维蛋白原家族的一员。近年来，有报道称FGL1是淋巴细胞激活基因3 （LAG3）的主要抑制配体。此外，FGL1-LAG3通路与程序性死亡1 (PD-1)/程序性死亡配体1 （PD-L1）通路具有协同作用，被认为是一个有前景的免疫治疗靶点。然而，猪FGL1 （sFGL1）尚未被鉴定，检测方法也缺乏。本研究从猪肝组织中扩增sFGL1基因，并将其插入原核表达载体pQE-30中。将重组质粒pQE30-sFGL1转染JM109感受态细胞。用异丙基-β-d-硫代半乳糖苷（IPTG）诱导表达重组sFGL1，纯化后的sFGL1作为抗原制备小鼠单克隆抗体（mAb）和兔多克隆抗体（pAb）。鉴定后，建立了灵敏特异检测sFGL1的双抗体夹心酶联免疫吸附试验（DAS-ELISA）。用抗sFGL1单抗捕获猪FGL1，然后用抗sFGL1兔单抗和酶标山羊抗兔IgG检测。构建的重组sFLG1- das - elisa检测限为35 pg/ml。此外，它与对照蛋白没有交叉反应性。采用建立的DAS-ELISA法检测prrsv阴性和阳性猪血清样本，计算公式为y=0.0735x+0.0737。结果表明，PRRSV感染显著提高了血清FGL1水平。本研究为进一步研究猪FGL1的功能作用提供了平台。
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.
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DOI：
10.1016/j.bbrc.2015.07.078
发表时间：
2015-09-18
影响因子：
3.1
作者：
Nayeb-Hashemi H;Desai A;Demchev V;Bronson RT;Hornick JL;Cohen DE;Ukomadu C
通讯作者：
Ukomadu C
影响因子：
--
作者：
Joller N;Kuchroo VK
通讯作者：
Kuchroo VK
DOI：
10.1016/j.bbrc.2007.11.069
发表时间：
2008-01-25
影响因子：
3.1
作者：
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Ukomadu, Chinweike
DOI：
10.1016/j.bbrc.2006.09.018
发表时间：
2006-11-10
影响因子：
3.1
作者：
Rijken, D. C.;Dirkx, S. P. G.;Leebeek, F. W. G.
通讯作者：
Leebeek, F. W. G.
影响因子：
24.5
作者：
Li, Chang-Yan;Cao, Chuan-Zeng;Yang, Xiao-Ming
通讯作者：
Yang, Xiao-Ming