Establishment and Preliminary Application of Double Antibody Sandwich ELISA Method for LECT2 of Ayu (Plecoglossus altivelis)
XU Li-Qiong1,2,3, SHI Yu-Hong2,3,*, CHEN Jiong1,2,3
1 State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China; 2 Laboratory of Biochemistry and Molecular Biology, School of Marine Science, Ningbo University, Ningbo 315211, China; 3 Key Laboratory of Aquacultral Biotechnology of Ministry of Education, School of Marine Science, Ningbo University, Ningbo 315832, China
Abstract：The expression level of leucocyte cell-derived chemotaxin 2 (LECT2) reflects the health status of fish. In this study, the recombinant PaLECT2 (rPaLECT2) protein, expressed by Pichia pastoris, was used to prepare anti-PaLECT2 antisera in mice (Mus musculus) and New Zealand white rabbits (Oryctolagus cuniculus). Then the antibodies were purified from antisera by Protein G affinity chromatography. The mouse anti-PaLECT2 antibody was used as the capture antibody and the rabbit anti-PaLECT2 antibody conjugated with horseradish peroxidase (HRP) as the detecting antibody. A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) method based on PaLECT2 protein was established after optimization. Finally, it was applied to detect the PaLECT2 in serum the Aeromonas hydrophila-infected ayu model and the prognosis model of antibiotic treatment. The results showed that the titer of PaLECT2 antibody in mice were 1∶12 800 and that of HRP-conjugated antibody in rabbits were 1∶25 600, respectively. The DAS-ELISA method for PaLECT2 used the optimal coating concentration of 8 μg/mL for the capture antibody and the best working dilution of 1∶1 600 for the detection antibody. The minimum detection limit of the method was 11.22 ng/mL with good specificity and stability. The protein concentration of PaLECT2 ((412±31.5) ng/mL) in the infected group was extremely significantly higher than that in the healthy group (54±7.2) ng/mL (P<0.001). The protein concentration of PaLECT2 in the treated group (225±56.1) ng/mL was extremely significantly lower than that in the infected group (P<0.01). In conclusion, the establishment of this method provides a more efficient and standard method for the quantitative measurement of PaLECT2 protein levels, which will be applied to the diagnosis and monitoring of ayu health status.
[1] 陈欢. 2022. 小鼠IL-1β和小鼠IL-12p40双抗体夹心ELISA方法的建立[D]. 硕士学位论文, 华中农业大学, 导师: 胡涌刚, pp. 12-51. (Chen H.2022. Double antibody sandwich ELISA methods for the detection of mouse IL-1β and IL-12p40[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Hu Y G, pp. 12-51.) [2] 程娜, 史雨红, 陈炯. 2014. 香鱼TGF-β1基因的克隆及其表达与鳗利斯顿氏菌感染的相关性[J]. 生物学杂志, 31(04): 1-5+28. (Cheng N, Shi Y H, Chen J. 2014. Cloning and expression of TGF-β1 gene in ayu [3] 李明云, 张呈念, 苗亮, 等. 2011. 香鱼嗜水气单胞菌染病后主要靶器官组织病理学观察[J].宁波大学学报(理工版), 24(03): 1-4. (Li M Y, Zheng C N, Miao L, et al.2011. Histopathological observation of main target organs after Aeromonas hydrophila infection in ayu (Plecoglossus altivelis)[J]. Journal of Ningbo University (Science and Technology), 24(03): 1-4.) [4] 李祥苏, 金星星, 王冬冬, 等. 2020. 血清LECT2检测对肝细胞癌诊断的意义[J]. 肝脏, 25(1): 7. (Li X S, Jin X X, Wang D D, et al. 2020. Significance of serum LECT2 detection in the diagnosis of hepatocellular carcinoma[J]. Liver, 25(1): 7.) [5] 李长红, 陈炯, 史雨红, 等. 2009. 宁海地区香鱼弧菌病病原菌鉴定[J]. 微生物学报, 49(7): 931-937. (Li C H, Chen J, Shi Y H, et al.2009. Characterization of Listonella anguillarum as the aetilogical agent of vibriosis occurred in cultured ayu (Plecoglossus altivelis) in Ninghai country, China[J]. Acta Microbiological Sinaica, 49(7): 931-937.) [6] 李长红, 陈炯, 史雨红.等. 2011. 香鱼抗凝血酶基因(AT) cDNA的克隆、序列分析及组织表达特征[J]. 农业生物技术学报, 19(01): 157-163. (Li C H, Chen J, Shi Y H, et al.2011. Cloning, sequence analysis and tissue expression characteristics of the cDNA of antithrombin gene (AT) in ayu (Plecoglossus altivelis)[J]. Journal of Agricultural Biotechnology, 19(01): 157-163.) [7] 马文静, 陈凯, 史雨红, 等. 2018. 香鱼Wap65-2在昆虫杆状病毒系统中的表达及其与补体C3的相互作用验证[J]. 农业生物技术学报, 26(10): 1762-1769. (Ma W J, Chen K, Shi Y H, et al.2018. Expression of ayu (Plecoglossus altivelis) Wap65-2 in the insect baculovirus system and its interaction with complement C3[J]. Journal of Agricultural Biotechnology, 26(10): 1762-1769.) [8] 沈广强, 陈炯, 史雨红, 等. 2013. 香鱼IL-12B基因序列及其表达与鳗利斯顿氏菌感染的相关性分析[J]. 中国细胞生物学学报, 35(09): 1314-1320. (Shen G Q, Chen J, Shi Y H, et al.2013. Correlation analysis of IL-12B gene sequence and expression in ayu (Plecoglossus altivelis) and Vibrio anguillarum infection[J]. Chinese Journal of Cell Biology, 35(09): 1314-1320.) [9] 王肖祎, 苏倩倩, 李恩扩, 等. 2014. 鸡白细胞介素 18 双抗体夹心ELISA检测方法的建立及初步应用[J]. 中国兽医学报, 34(1): 123-126. (Wang X Y, Su Q Q, Li E K, et al.2014. Establishment and preliminary application of double antibody sandwich ELISA for detection of chicken interleukin-18[J]. Chinese Veterinary Journal, 34(1): 123-126.) [10] 吴美英, 艾艳萍, 曹艳, 等. 2012. 双抗体夹心ELISA定量检测重组人干扰素αlb方法的建立与评价[J]. 中华实验和临床病毒学杂志, 26(6): 489-491. (Wu M Y, Ai Y P, Cao Y, et al.2012. Establishment and evaluation of double antibody sandwich ELISA for quantitative detection of recombinant human IFN-αlb[J]. Chinese Journal of Experimental and Clinical Virology, 26(6): 489-491.) [11] 杨红艳, 陈炯, 史雨红, 等. 2009. 香鱼LECT2基因的克隆及其组织表达差异[J]. 细胞生物学杂志, 31(2): 277-280. (Yang H Y, Chen J, Shi Y H, et al.2009. Cloning and tissue expression difference of LECT2 gene in ayu (Plecoglossus altivelis)[J]. Journal of Cell Biology, 31(2): 277-280.) [12] 周昊. 2018. 草鱼三种细胞因子的表达载体构建及白介素-10 DAS-ELISA检测方法的建立[D]. 硕士学位论文, 安徽农业大学, 导师: 李槿年, pp. 1-32. (Zhou H.2018. Construction of expression vectors for three cytokines in grass carp and establishment DAS-ELISA assay for detecting interleukin-10[D]. Thesis for M.S., Anhui Agricultural University, Supervisor: Li J N, pp. 1-32.) [13] Berraondo P, Sanmamed M F, Ochoa M C, et al.2019. Cytokines in clinical cancer immunotherapy[J]. British Journal of Cancer, 120(1): 6-15. [14] Chen J, Shi Y H, Hu H Q, et al.2009 Apolipoprotein AI, a hyperosmotic adaptation-related protein in ayu (Plecoglossus altivelis)[J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 152(2): 196-201. [15] Chen J, Chen Q, Lu X J, et al.2014. LECT2 improves the outcomes in ayu with Vibrio anguillarum infection via monocytes/macrophages[J]. Fish & Shellfish Immunology, 41(2): 586-592. [16] Fan B, Sun J, Zhu L, et al.2020. Development of a novel double antibody sandwich quantitative enzyme-linked immunosorbent assay for detection of porcine Epidemic diarrhea virus antigen[J]. Frontiers in Veterinary Science, 7: 540248. [17] Fujiki K, Shin D H, Nakao M, et al.2000. Molecular cloning of carp (Cyprinus carpio) leucocyte cell-derived chemotaxin 2, glia maturation factor beta, CD45 and lysozyme C by use of suppression subtractive hybridisation[J]. Fish & Shellfish Immunology, 10(7): 643-650. [18] Greenow K R, Zverev M, May S, et al.2018. Lect2 deficiency is characterised by altered cytokine levels and promotion of intestinal tumourigenesis[J]. Oncotarget, 9(92): 36430. [19] Gu Y, Guo X, Liu P, et al.2021. Preparation of ribosomal protein S14 polyclonal antibody in broiler pulmonary artery: Its application in broiler ascites syndrome[J]. International Journal of Biological Macromolecules, 193: 328-336. [20] Hu Y Z, Ma Z Y, Wu C S, et al.2022. LECT2 is a novel antibacterial protein in vertebrates[J]. The Journal of Immunology, 208(8): 2037-2053. [21] Hwang H J, Jung T W, Hong H C, et al.2015. LECT2 induces atherosclerotic inflammatory reaction via CD209 receptor-mediated JNK phosphorylation in human endothelial cells[J]. Metabolism, 64(9): 1175-1182. [22] Ikeda D, Ageta H, Tsuchida K, et al.2013. iTRAQ-based proteomics reveals novel biomarkers of osteoarthritis[J]. Biomarkers, 18(7): 565-572. [23] Jung T W, Chung Y H, Kim H C, et al.2018. LECT2 promotes inflammation and insulin resistance in adipocytes via P38 pathways[J]. Journal of Molecular Endocrinology, 61(1): 37-45. [24] Kokkinos P A, Kazantzi A, Sfyroera G, et al.2005. Molecular cloning of leukocyte cell-derived chemotaxin 2 in rainbow trout[J]. Fish & Shellfish Immunology, 18(5): 371-380. [25] Lan F, Misu H, Chikamoto K, et al.2014. LECT2 functions as a hepatokine that links obesity to skeletal muscle insulin resistance[J]. Diabetes, 63(5): 1649-1664. [26] Li Q, Zhang Z, Fan W, et al.2021a. LECT2 protects Nile tilapia (Oreochromis niloticus) against Streptococcus agalatiae infection[J]. Frontiers in Immunology, 12: 667781. [27] Li X, Lin P, Tao Y, Jiang X, et al.2021b. LECT 2 antagonizes FOXM1 signaling via inhibiting MET to retard PDAC progression[J]. Frontiers in Cell and Developmental Biology, 9: 661122. [28] Long C, Lai Y, Li T, et al.2020. Cigarette smoke extract modulates Pseudomonas aeruginosa bacterial load via USP25/HDAC11 axis in lung epithelial cells[J]. The American Journal of Physiology-Lung Cellular and Molecular Physiology, 318(2): L252-L263. [29] Lu X J, Chen J, He Y Q, et al.2013a. Molecular characterization of an IL-1β gene from ayu, Plecoglossus altivelis[J]. Fish & Shellfish Immunology, 34(5): 1253-1259. [30] Lu X J, Chen J, Yu C H, et al.2013b. LECT2 protects mice against bacterial sepsis by activating macrophages via the CD209a receptor[J]. Journal of Experimental Medicine, 210(1): 5-13. [31] Ma W J, Shi Y H, Chen J.2020. Ayu (Plecoglossus altivelis) CD46 isoforms protect the cells from autologous complement attack[J]. Fish & Shellfish Immunology, 102: 267-275. [32] Mathew J A, Guo Y X, Goh K P, et al.2002. Characterisation of a monoclonal antibody to carp IL-1β and the development of a sensitive capture ELISA[J]. Fish & Shellfish Immunology, 13(2): 85-95. [33] Nishimori E, Kita-Tsukamoto K, Wakabayashi H.2000. Pseudomonas plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis[J]. International Journal of Systematic and Evolutionary Microbiology, 50(1): 83-89. [34] Okumura A, Unoki-Kubota H, Matsushita Y, et al.2013. Increased serum leukocyte cell-derived chemotaxin 2 (LECT2) levels in obesity and fatty liver[J]. BioScience Trends, 7(6): 276-283. [35] Paweska J T, Burt F J, Swanepoel R.2005. Validation of IgG-sandwich and IgM-capture ELISA for the detection of antibody to Rift Valley fever virus in humans[J]. Journal of Virological Methods, 124(1-2): 173-181. [36] Persson S, Al-Shuweli S, Yapici S, et al.2015. Identification of clinical Aeromonas species by rpoB and gyrB sequencing and development of a multiplex PCR method for detection of Aeromonas hydrophila, A. caviae, A. veronii, and A. media[J]. Journal of Clinical Microbiology, 53(2): 653-656. [37] Shen Y, Cao M, Tang S, et al.2020. Genomic and functional characterization of the lect2 gene from Siniperca chuatsi[J]. Fish & Shellfish Immunology, 107(Pt A): 146-155. [38] Takata N, Ishii K, Takayama H, et al.2021. LECT2 as a hepatokine links liver steatosis to inflammation via activating tissue macrophages in NASH[J]. Scientific Reports, 11(1): 555. [39] Tanisawa K, Taniguchi H, Sun X, et al.2017. Visceral fat area is a strong predictor of leukocyte cell-derived chemotaxin 2, a potential biomarker of dyslipidemia[J]. PLOS ONE, 12(3): e0173310. [40] Wang W, Li J, Fan B, et al.2021. Development of a novel double antibody sandwich ELISA for quantitative detection of porcine deltacoronavirus antigen[J]. Viruses, 13(12): 2403. [41] Wu X Y, Xiong J B, Fei C J, et al.2022. Prior exposure to ciprofloxacin disrupts intestinal homeostasis and predisposes ayu (Plecoglossus altivelis) to subsequent Pseudomonas plecoglossicida-induced infection[J]. Zoological Research, 43(4): 648-665. [42] Xie Y, Fan K W, Guan S X, et al.2022. LECT2: A pleiotropic and promising hepatokine, from bench to bedside[J]. Journal of Cellular and Molecular Medicine, 26(13): 3598-3607. [43] Xu M, Xu H H, Lin Y, et al.2019. LECT2, a ligand for tie1, plays a crucial role in liver fibrogenesis[J]. Cell, 178(6): 1478-1492. [44] Zhang X, Li M, Zhang B, et al.2016. Development of a sandwich ELISA for EHEC O157: H7 intimin γ1[J]. PLOS ONE, 11(9): e0162274. [45] Zhang Z, Niu J, Li Q, et al.2021. HMG20A from Nile tilapia (Oreochromis niloticus) involved in the immune response to bacterial infection[J]. Fish & Shellfish Immunology, 119: 499-507.
