Establishment of a Double-antibody Sandwich ELISA for Detection of S100A8 in Forest Musk Deer (Moschus berezovskii)
SUO Li-Juan1,2,3, BIAN Kun1,2,3, LI Fei-Ran1,2,3, YANG Chao1,2,3, TANG Jie1,2,3,*
1 Shaanxi Key Laboratory of Qinling Ecological Security, Shaanxi Institute of Zoology, Xi'an 710032, China; 2 Baoji City Forest Musk Engineering Technology Research Center, Feng County 721700, China; 3 Foping county forest musk expert workstation, Foping County 723400, China
Abstract：The forest musk deer (Moschus berezovskii), a first-class protected animal in China, has musk secreted by its male that holds significant medicinal value. The S100A8 protein, as a member of the calcium - binding protein family, plays a crucial role in the inflammatory response. Therefore, to meet the health - monitoring needs in the artificial breeding of forest musk deer, a double-antibody sandwich ELISA for S100A8 was established to detect S100A8 in the serum samples of forest musk deer.In this study, the prokaryotic expression vector pET28a-B2M was constructed to obtain the S100A8 recombinant protein. Rabbit-derived polyclonal antibodies were obtained by multi-point injection into New Zealand white rabbits (Oryctolagus cuniculus). Mouse-derived S100A8 monoclonal antibodies were prepared through hybridoma technology and used as capture antibodies. The horseradish peroxidase (HRP) labeled rabbit-derived S100A8 polyclonal antibodies were used as detection antibodies. A double-antibody sandwich ELISA quantitative detection method for S100A8 in musk deer was established, and the antibody concentration, optimal coating conditions of the capture antibody and incubation conditions were optimized. The established ELISA method was used to specifically detect 31 serum samples.The results showed that the concentration of the recombinant protein obtained by Ni2+ column affinity chromatography was 5 mg/mL, with a purity of over 85%. Indirect ELISA indicated that the titers of both mouse and HRP-rabbit anti-S100A8 antibodies were higher than 1∶50 000. When the concentration of the capture antibody was 4 μg/mL and the dilution ratio of the detection antibody was 1∶2 000, these were the optimal coating concentration of the capture antibody and the working concentration of the detection antibody. The minimum sensitivity of the double-antibody sandwich ELISA method established in this study was 15.625 ng/mL, and it could specifically distinguish between inflammatory samples and normal samples. A double-antibody sandwich ELISA detection method was successfully established to achieve the quantitative detection of S100A8 protein in samples. It is expected to be applied to comprehensively evaluate the health status of forest musk deer and provide support for the early diagnosis and treatment of diseases.
索丽娟, 边坤, 李斐然, 杨超, 唐婕. 林麝S100A8双抗体夹心ELISA检测方法的建立[J]. 农业生物技术学报, 2025, 33(10): 2288-2298.
SUO Li-Juan, BIAN Kun, LI Fei-Ran, YANG Chao, TANG Jie. Establishment of a Double-antibody Sandwich ELISA for Detection of S100A8 in Forest Musk Deer (Moschus berezovskii). 农业生物技术学报, 2025, 33(10): 2288-2298.
[1] 戴晓阳, 杨营, 王建明, 等. 2012. 林麝血液生理生化指标的测定[J]. 四川动物, 31(02): 256-258. (Dai X Y, Yang Y, Wang J M, et al.2012. Blood physiological and biochemical characterization of forest musk deer[J]. Sichuan Journal of Zoology, 31(02): 256-258.) [2] 方普. 2022. 血清S100A8作为社区获得性肺炎患者的早期诊断生物标志物[D]. 硕士学位论文, 安徽医科大学, 导师: 陆友金, pp. 32. (Fang P. 2022. Serum S100A8 as an early diagnostic biomarker in patients with community acquired pneumonia[J]. Thesis for M.S., Anhui Medical University, Supervisor: Lu Y J, pp. 32.) [3] 韩金洁, 冯梦瑶, 房军洋, 等. 2024. 林麝出血性肺炎病原菌的分离鉴定和菌影疫苗的制备[J]. 中国兽医杂志, 60(03): 27-34. (Han J J, Feng M Y, Fang J Y, et al.2024. Isolation and identification of pathogenic bacteria causing hemorrhagic pneumonia in moschus berezovsky and preparation of bacterial ghost vaccine[J]. Chinese Journal of Veterinary Medicine, 60(03): 27-34.) [4] 刘春燕. 2008. 林麝Moschus berezovskii养殖种群死亡原因及其生命生理特征研究[D]. 硕士学位论文, 华东师范大学, 导师: 徐宏发, pp. 61. (Liu C Y. 2008. A study on reasons of death and physiological features for breeding forest musk deer (Moschus berezovskii)[D]. Thesis for M.S., Anhui Medical University, Supervisor: Lu Y J, pp. 61) [5] 刘玉太, 金学林, 赵泽明, 等. 2021. 一例林麝支气管败血波氏杆菌性肺炎的诊断[J]. 特种经济动植物, 24(08): 26-30. (Liu Y T, Jin X L, Zhao Z M, et al.2021. Diagnosis of a case of forest musk deer bronchial septicemia Bordetella pneumonia[J]. Special Economic Animals and Plants, 24(08): 26-30.) [6] 苗朝悦, 杜乐, 王佳琦, 等. 2023. 重组蛋白质在大肠杆菌体系中的可溶性表达策略[J]. 中国生物工程杂志, 43(09): 33-45. (Miao C Y, Du L, Wang J Q, et al.2023. Soluble expression strategies for production of recombinant proteins in Escherichia coli[J]. China Biotechnology,43(09): 33-45.) [7] 彭红元, 陈伟才, 张修月. 2010. 麝的分类研究概述[J]. 玉林师范学院学报, 31(02): 66-72. (Peng H Y, Chen W C, Zhang X Y.2010. A review on the classification of musk deer[J]. Journal of Yulin Normal University (Natural Science), 31(02): 66-72.) [8] 祁佳丽. 2022. 凤县人工养殖林麝常见疾病调查及呼吸道病原菌的检测分离[D]. 硕士学位论文, 西北农林科技大学, 导师: 郭抗抗, 吕长荣, pp. 17-39. (Qi J L.2022. Investigation of common diseases of artificial breeding forest musk deer and detection and isolation of respiratory pathogens in Fengxian County[D]. Thesis for M.S., Northwest Agriculture and Forestry Technology University, Supervisor: Guo K K, Lu C R, pp. 17-39.) [9] 秦逸飞, 陈蓉, 施振旦. 2024. 鹅VDR基因克隆及其多克隆抗体制备与鉴定[J]. 中国畜牧兽医, 51(03): 1194-1202. (Qin Y F, Chen R, Shi Z D.2024. Cloning of VDR gene and preparation and identification of its polyclonal antibody in geese[J]. China Animal Husbandry & Veterinary Medicine, 51(03): 1194-1202.) [10] 王芳, 苏娟. 2021. 血清S100A8/A9蛋白在类风湿关节炎中的研究进展[J]. 中文科技期刊数据库(全文版)医药卫生, (6): 373-375. (Wang F, Su J. 2021. Research progress of serum S100A8 / A9 protein in rheumatoid arthritis[J]. Chinese Journal of Integrative Nursing, (6): 373-375.) [11] 许萌萌, 黄萌萌, 牛鑫鑫, 等. 2024. 鸡pro-IL-1β表达、多克隆抗体制备与初步应用[J]. 中国家禽, 46(10): 89-96. (Xu M M, Huang M M, Niu X X, et al.2024. Expression, polyclonal antibody preparation and it's preliminary application of chicken pro-IL-1β[J]. China Poultry,46(10): 89-96.) [12] 闫丽. 2023. 猪源S100A8、S100A9蛋白抑菌活性探究及单克隆抗体的制备与应用[D]. 硕士学位论文, 扬州大学,导师: 夏芃芃, 周明旭, pp. 29-39. (Li Y.2023.Study on antibacterial activity of porcine S100A8 and S100A9 proteins and the production and application of their monoclonal antibodies[D]. Thesis for M.S., Yangzhou University, Supervisor: Xia P P, Zhou M X, pp. 29-39.) [13] Ascoli C A, Aggeler B.2018. Overlooked benefits of using polyclonal antibodies[J]. Biotechniques, 65(3): 127-136. [14] Chen H, Lunney J K, Cheng L, et al.2011. Porcine S100A8 and S100A9: Molecular characterizations and crucial functions in response to Haemophilus parasuis infection[J]. Developmental & Comparative Immunology, 35(4):490-500. [15] Choi I Y, Gerlag D M, Herenius M J, et al.2015. MRP8/14 serum levels as a strong predictor of response to biological treatments in patients with rheumatoid arthritis[J]. Annals of the Rheumatic Diseases, 74(3): 499-505. [16] Crowe L A N, McLean M, Kitson S M, et al.2019. S100A8 & S100A9: Alarmin mediated inflammation in tendinopathy[J]. Scientific Reports, 9(1): 1463. [17] Feng H, Wang L, Cao F, et al.2023. Forest musk deer (Moschus berezovskii) in China: Research and protection[J]. Journal of Vertebrate Biology, 72: 22067. [18] Foell D, Frosch M, Sorg C, et al.2004. Phagocyte-specific calcium-binding S100 proteins as clinical laboratory markers of inflammation[J]. Clinica Chimica Acta, 344(1): 37-51. [19] Aleksandra H, Kristina S, Per V, et al.2020. Calprotectin, a new biomarker for diagnosis of acute respiratory infections[J]. Scientific Reports, 10(1): 4208. [20] Kim J P, Yun H, Kim E J, et al.2023. Generation of a novel monoclonal antibody against inflammatory biomarker S100A8 using hybridoma technology[J]. Biotechnology Letters, 45(5-6): 589-600. [21] Kitagori K, Oku T, Wakabayashi M, et al.2023. Expression of S100A8 protein on B cells is associated with disease activity in patients with systemic lupus erythematosus[J]. Arthritis Research and Therapy, 25(1): 76. [22] Lagasse E, Clerc R G.1988. Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation[J]. Molecular Cell Biology, 8(6): 2402-2410. [23] Moore B W, McGregor D.1965. Chromatographic and electrophoretic fractionation of soluble proteins of brain and liver[J]. Journal of Biological Chemistry, 240: 1647-1653. [24] Müller I, Vogl T, Kühl U, et al.2020. Serum alarmin S100A8/S100A9 levels and its potential role as biomarker in myocarditis[J]. ESC Heart Fail, 7(4): 1442-1451. [25] Murdoch C C, Skaar E P.2022. Nutritional immunity: The battle for nutrient metals at the host-pathogen interface[J]. Nature Reviews Microbiology, 20(11): 657-670. [26] Nakazawa M, Mukumoto M, Miyatake K.2010. Production and purification of polyclonal antibodies[J]. Methods in Molecular Biology, 657: 63-74. [27] Ometto F, Friso L, Astorri D, et al.2017. Calprotectin in rheumatic diseases[J]. Experimental Biology and Medicine (Maywood, N.J.), 242(8): 859-873. [28] Parray H A, Shukla S, Samal S, et al.2020. Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives[J]. International Immunopharmacology, 85: 106639. [29] Posner J, Barrington P, Brier T, et al.2019. Monoclonal antibodies: Past, present and future[J]. Handbook of Experimental Pharmacology, 260: 81-141. [30] Tang J, Suo L, Li F, et al.2022. Transcriptome profiling of lung immune responses potentially related to acute respiratory distress syndrome in forest musk deer[J]. BMC Genomics, 23(1): 701. [31] Wang S, Song R, Wang Z, et al.2018. S100A8/A9 in Inflammation[J]. Frontiers in Immunology, 9: 1298. [32] Xia P, Ma X, Yan L, et al.2024. Generation and application of monoclonal antibodies against porcine S100A8, S100A9, and S100A12 proteins using hybridoma technology[J]. International Journal of Molecular Sciences, 25(2): 1029. [33] Yagami H, Kato H, Tsumoto K, et al.2013. Monoclonal antibodies based on hybridoma technology[J]. Pharmaceutical Patent Analyst, 2(2): 249-263. [34] Zackular J P, Chazin W J, Skaar E P.2015. Nutritional immunity: S100 proteins at the host-pathogen interface[J]. Journal of Biological Chemistry, 290(31): 18991-18998.
