牛血清淀粉样蛋白A时间分辨荧光免疫层析检测试剂盒研制

doi:10.16431/j.cnki.1671-7236.2020.04.025

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (4): 1191-1198.doi: 10.16431/j.cnki.1671-7236.2020.04.025

牛血清淀粉样蛋白A时间分辨荧光免疫层析检测试剂盒研制

吴萌¹, 魏治静¹, 樊一明², 崔晨光³, 李双⁴, 董维亚⁵

1. 河北省科学院生物研究所, 石家庄 050081;
2. 河北农业大学动物医学院(中兽医学院), 保定 071000;
3. 河北省行唐县畜产品质量监测站, 石家庄 050600;
4. 石家庄君乐宝乳业有限公司, 石家庄 050221;
5. 河北省动物疫病预防控制中心, 石家庄 050000

收稿日期:2019-09-27 出版日期:2020-04-20 发布日期:2020-04-17
通讯作者: 吴萌 E-mail:biobang@sina.com
作者简介:吴萌(1975-),男,河北石家庄人,硕士,副研究员,研究方向:免疫细胞学
基金资助:
河北省重点研发计划项目"两种急性期蛋白用于奶牛乳房炎诊断的免疫快速检测技术研究"（19222814D）；河北省科学院科技计划项目"奶牛隐性乳腺炎诊断标志物抗体研制及定量检测技术研究"（19302）

Development of a Time-resolved Fluorescence Immunochromatography Assay Kit for Detection of Bovine Serum Amyloid A

WU Meng¹, WEI Zhijing¹, FAN Yiming², CUI Chenguang³, LI Shuang⁴, DONG Weiya⁵

1. Biology Institute, Hebei Academy of Sciences, Shijiazhuang 050081, China;
2. College of Veterinary Medicine(Traditional Chinese Veterinary Medicine), Hebei Agricultural University, Baoding 071000, China;
3. Animal Products Quality Monitoring Station of Xingtang County, Hebei Province, Shijiazhuang 050600, China;
4. Shijiazhuang Junlebao Dairy Co., Ltd., Shijiazhuang 050221, China;
5. Hebei Animal Epidemic Prevention and Control Center, Shijiazhuang 050000, China

Received:2019-09-27 Online:2020-04-20 Published:2020-04-17

摘要/Abstract

摘要： 本研究旨在研制一种牛血清淀粉样蛋白A（SAA）时间分辨荧光免疫层析定量检测试剂盒，用于牛奶中SAA含量的临床快速检测。采用双抗体夹心法结合荧光免疫层析技术，在结合垫上固定荧光微球标记的抗SAA单克隆抗体及荧光微球标记的鸡IgY的混合物，在硝酸纤维素膜的检测区包被另一株抗SAA单克隆抗体，在硝酸纤维素膜的质控区包被山羊抗鸡IgY。经抗体原料筛选及荧光微球标记抗体的工艺优化后，绘制标准曲线并对试剂盒的空白限、精密度、稳定性及样本测试性能进行初步评估。结果显示，Medix SAA-2单克隆抗体包被与YBX SAA-3单克隆抗体标记为最适抗体配对原料。荧光微球标记抗体的工艺中，荧光微球与抗体的质量投料比为40∶1、偶联剂与荧光微球羧基摩尔比为2∶1的条件为最优组合。试剂盒标准曲线的四参数拟合曲线方程为y=（1.03947-0.00182）/[1＋（x/12.08222）×（-0.84692）]＋0.00182，线性相关系数R²=0.9997。研制的牛SAA检测试剂盒空白限为0.052 mg/L。精密度测试结果显示，批内变异系数 < 15%，批间变异系数 < 20%。室温稳定性试验表明，试剂盒在室温密封存放6个月的荧光T/C值相对跌幅约15%。自制试剂盒与上海蓝基试剂盒的样本对比测试相关系数R²为0.97。综上所述，本试验研制的试剂盒具有操作简便、灵敏度高、成本低廉等优点，能满足临床测定需求，可作为一种新型牛SAA检测的快捷、准确的检测手段。

关键词: 牛血清淀粉样蛋白A; 乳腺炎; 荧光免疫层析; 定量检测

Abstract: The purpose of this study was to develop a time-resolved fluorescence immunochromatography kit for clinical rapid detection of bovine serum amyloid A (SAA) content in milk.By using double antibody sandwich method and fluorescence immunochromatography detection technology,the immunochromatography detection kit was manufactured.The SAA monoclonal antibody used for labeling was labeled with fluorescent microspheres,and chicken IgY was labeled with fluorescent microspheres,too.Then the above two kinds of fluorescent microspheres labeled antibody mixture was fixed on the binding pad.The SAA monoclonal antibody used for coating was coated on the detection area of nitrocellulose membrane,and goat anti-chicken IgY was coated on the quality control area of nitrocellulose membrane.The standard curve of the kit was drawn after screening of antibody pairing materials and optimization of the process of fluorescent microsphere labeled antibody.Besides,the blank limit,precision,stability and sample testing performance of the kit were preliminarily evaluated.The results showed that,the Medix SAA-2 monoclonal antibody as coating antibody and YBX SAA-3 monoclonal antibody as labeling antibody was the optimum antibody pairing materials.In the process of labeling antibody with fluorescent microsphere,the optimal results were that the mass ratio of fluorescent microspheres to antibodies was 40∶1,and the carboxyl molar ratio of coupling agent to fluorescent microspheres was 2∶1.The four-parameter fitting curve equation of the standard curve of the kit was y=(1.03947-0.00182)/[1＋(x/12.08222)×(-0.84692)]＋0.00182,the linear correlation coefficient was R²=0.9997.The blank limit of the developed bovine SAA detection kit was 0.052 mg/L.Precision test result showed that,the intra-assay coefficient of variation was < 15%,and the inter-assay coefficient of variation was < 20%.The room temperature stability test showed that,the fluorescence T/C value of the sealed kit stored at room temperature for 6 months decreased by about 15%.The correlation coefficient R² of self-made kit and Shanghai Blue Gene kit was 0.97.In summary,the developed kit had the advantages of simple operation,high sensitivity and low cost,which could meet the needs of clinical determination,and provided a new type of fast and accurate detection method for bovine SAA detection.

Key words: bovine serum amyloid A; mastitis; fluorescence immunochromatography; quantitative detection

中图分类号:

S858.23

吴萌, 魏治静, 樊一明, 崔晨光, 李双, 董维亚. 牛血清淀粉样蛋白A时间分辨荧光免疫层析检测试剂盒研制[J]. 中国畜牧兽医, 2020, 47(4): 1191-1198.

WU Meng, WEI Zhijing, FAN Yiming, CUI Chenguang, LI Shuang, DONG Weiya. Development of a Time-resolved Fluorescence Immunochromatography Assay Kit for Detection of Bovine Serum Amyloid A[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(4): 1191-1198.

参考文献

[1] 尹柏双,付连军,郝景锋,等.我国奶牛隐性乳房炎诊断方法的研究现状[J].黑龙江畜牧兽医,2014,6:54-56. YIN B S,FU L J,HAO J F,et al.Current status of diagnostic methods for subclinical mastitis in dairy cows in China[J].Heilongjiang Animal Science and Veterinary Medicine,2014,6:54-56.(in Chinese)
[2] 刘来钦.奶牛乳房炎及其防治[J].畜牧兽医科技信息,2016,9:67-68. LIU L Q.Dairy cow mastitis and its prevention and treatment[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2016,9:67-68.(in Chinese)
[3] 韩旭,武瑞,孙东波.奶牛乳房炎主要病原菌基因检测技术研究进展[J].中国畜牧兽医,2012,39(3):230-232. HAN X,WU R,SUN D B.Advances in the research on gene detection technology of main pathogenic bacteria of cow mastitis[J].China Animal Husbandry&Veterinary Medicine,2012,39(3):230-232.(in Chinese)
[4] 王丹,杨峰,李新圃,等.奶牛乳房炎病原菌诊断技术研究进展[J].中国畜牧兽医,2017,44(6):1811-1817. WANG D,YANG F,LI X P,et al.Research progress on diagnosis technology of dairy cow mastitis pathogens[J].China Animal Husbandry&Veterinary Medicine,2017,44(6):1811-1817.(in Chinese)
[5] 郭英,滕达,王秀敏,等.抗菌肽NZ2114对奶牛乳房炎源金黄色葡萄球菌细胞膜、基因组和耐药性的影响[J].中国畜牧兽医,2019,46(4):1181-1190. GUO Y,TENG D,WANG X M,et al.Effect of antimicrobial peptide NZ2114 on cell membrane,genome and antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis milk[J].China Animal Husbandry&Veterinary Medicine,2019,46(4):1181-1190.(in Chinese)
[6] 高原,张博,姚伟.奶牛乳房炎实验室诊断技术研究进展[J].现代畜牧兽医,2018,4:54-59. GAO Y,ZHANG B,YAO W.Research progress in diagnostic techniques of dairy cow mastitis in laboratory[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2018,4:54-59.(in Chinese)
[7] UHLAR C M,WHITEHEAD A S.Serum amyloid A,the major vertebrate acute-phase reactant[J].European Journal of Biochemistry,1999,265(2):501-523.
[8] ECKERSALL P D,YOUNG F J,MCCOMB C,et al.Acute phase proteins in serum and milk from dairy cows with clinical mastitis[J].Veterinary Record,2001,148(2):35-41.
[9] GRONLUND U,HULTEN C,ECKERSALL P D,et al.Haptoglobin and serum amyloid A in milk and serum during acute and chronic experimentally induced Staphylococcus aureus mastitis[J].Journal of Dairy Research,2003,70(4):379-386.
[10] 张思学,彭立颖,王洪伟,等.不同程度奶牛乳腺炎乳中血清淀粉样蛋白A的变化[J].中国兽医杂志,2011,47(12):38-39. ZHANG S X,PENG L Y,WANG H W,et al.Changes of serum amyloid A in dairy cows with different degrees of mastitis[J].Chinese Journal of Veterinary Medicine,2011,47(12):38-39.(in Chinese)
[11] 薛永康,闫磊,张震,等.乳清急性期蛋白、乳酶作为奶牛隐性乳腺炎诊断指标的研究[J].中国兽医学报,2018,38(8):1592-1597. XUE Y K,YAN L,ZHANG Z,et al.Evaluation of APP and lactase as indicatrix for the diagnosis of sub-clinical bovine mastitis[J].Chinese Journal of Veterinary Science,2018,38(8):1592-1597.(in Chinese)
[12] MCDONALD T L,WEBER A,SMITH J W.A monoclonal antibody sandwich immunoassay for serum amyloid A (SAA) protein[J].Journal of Immunological Methods,1991,144(2):149-155.
[13] ECKERSALL P D,YOUNG F J,NOLAN A M,et al.Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis[J].Journal of Dairy Science,2006,89(5):1488-1501.
[14] O'MAHONY M C,HEALY A M,HARTE D,et al.Milk amyloid A:Correlation with cellular indices of mammary inflammation in cows with normal and raised serum amyloid A[J].Research in Veterinary Science,2006,80(2):155-161.
[15] SZCZUBIAL M,DABROWSKI R,KANKOFER M,et al.Concentration of serum amyloid A and activity of ceruloplasmin in milk from cows with clinical and subclinical mastitis[J].Bulletin of the Veterinary Institute in Pulawy,2008,52:391-395.
[16] THOMAS F C,WATERSTON M,HASTIE P,et al.The major acute phase proteins of bovine milk in a commercial dairy herd[J].BMC Veterinary Research,2015,11(1):207-217.
[17] THOMAS F C,WATERSTON M,HASTIE P,et al.Early post parturient changes in milk acute phase proteins[J].Journal of Dairy Research,2016,83(3):352-359.
[18] 杨德英.奶牛乳房炎对乳酶活性、急性期蛋白含量的影响及其作为隐性乳房炎诊断指标的研究[D].成都:四川农业大学,2009. YANG D Y.Effects of bovine mastitis on activity of milk enzymes,content of acute phase proteins and as potential indicator of cow's[D].Chengdu:Sichuan Agricultural University,2009.(in Chinese)
[19] 薛永康.奶牛乳腺炎快速诊断技术的应用与隐性乳腺炎诊断指标的探索[D].郑州:河南农业大学,2017. XUE Y K.Application on rapid diagnosis of bovine mastitis and exploration on diagnostic indicators of sub-clinical bovine mastitis[D].Zhengzhou:Henan Agricultural University,2017.(in Chinese)
[20] 杭建峰,吴英松,李明.时间分辨荧光免疫分析的研究进展及应用[J].热带医学杂志,2004,4(3):340-343. HANG J F,WU Y S,LI M.Research progress and application of time-resolved fluorescence immunoassay[J].Journal of Tropical Medicine,2004,4(3):340-343.(in Chinese)
[21] 王云龙,米亚双,李玉林,等.CA19-9时间分辨荧光免疫层析检测方法的建立[J].生物工程学报,2018,34(6):1012-1018. WANG Y L,MI Y S,LI Y L,et al.Establishment of time-resolved fluorescence immunochromatographic assay for detection of carbohydrate antigen 19-9[J].Chinese Journal of Biotechnology,2018,34(6):1012-1018.(in Chinese)
[22] FANG S,ZHANG Y Z,LIU X,et al.Development of a highly sensitive time-resolved fluoroimmunoassay for the determination of trace salbutamol in environmental samples[J].Science of the Total Environment,2019,679:359-364.
[23] 尤震晨,王夕虎,葛建军,等.乳房及肢蹄洁净度对荷斯坦奶牛体细胞数的影响[J].中国畜牧兽医,2019,46(8):2390-2396. YOU Z C,WANG X H,GE J J,et al.Effect of udder and leg cleanliness on somatic cell count in Holstein dairy cows[J].China Animal Husbandry&Veterinary Medicine,2019,46(8):2390-2396.(in Chinese)
[24] 伍涛,赵兴绪.乳房炎奶牛乳汁体细胞数与乳汁HP、SAA和AGP水平关系的研究[J].甘肃农业大学学报,2008,43(6):19-24. WU T,ZHAO X X.Correlation of somatic cell count with the haptoglobin,serum amyloid A and α1-acid glycoprotein concentrations in composite milk from dairy cows with mastitis[J].Journal of Gansu Agricultural University,2008,43(6):19-24.(in Chinese)
[25] NIELSEN B H,JACOBSEN S,ANDERSEN P H,et al.Acute phase protein concentrations in serum and milk from healthy cows,cows with clinical mastitis and cows with extramammary inflammatory conditions[J].Veterinary Record,2004,154(12):361-365.
[26] LINDMARK-MANSSON H,BRANNING C,ALDEN G,et al.Relationship between somatic cell count,individual leukocyte populations and milk components in bovine udder quarter milk[J].International Dairy Journal,2006,16(7):717-727.
[27] BOCHNIARZ M,ZDZISINSKA B,WAWRON W,et al.Milk and serum IL-4,IL-6,IL-10,and amyloid A concentrations in cows with subclinical mastitis caused by coagulase-negative staphylococci[J].Journal of Dairy Science,2017,100(12):9674-9680.

牛血清淀粉样蛋白A时间分辨荧光免疫层析检测试剂盒研制

Development of a Time-resolved Fluorescence Immunochromatography Assay Kit for Detection of Bovine Serum Amyloid A

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