伏马毒素B_1单克隆抗体的制备及多种免疫学检测方法的建立
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摘要
伏马毒素(Fumonisins)是由串珠镰刀菌产生的真菌毒素,目前已知有28种衍生物,其中以伏马毒素B1(FB1)最为普遍,FB1能够对玉米及其制品造成污染,以谷物为原料的一些产品中如面条、啤酒、调味品、动物饲料,甚至在芦笋中均检测到了FB1。FB1毒性在伏马毒素中最强,研究也最为深入,对多种动物有较严重的毒理作用。研究表明伏马毒素B1可引发马的白脑软化症,猪的肺水肿综合症,此外,还可诱发人类的食道癌和肝癌、胃癌等疾病,对畜牧业和人类的健康构成危害。本研究在获得完全抗原,制备抗FB1单克隆抗体的基础上,建立了针对FB1的间接竞争ELISA、间接竞争磁酶免疫法及化学发光ELISA等多种检测方法,以期开发出适合不同检测样本,在不同条件下使用的特异性敏感的检测方法。
FB1是一种不具备免疫原性的半抗原小分子,必须通过与载体蛋白的偶联获得免疫原性。本文通过戊二醛法将半抗原FB1与载体蛋白钥孔血蓝蛋白(KLH)和蛋白卵清白蛋白(OVA)分别偶联成为完全抗原KLH-FB1与OVA-FB1,OVA-FB1作为包被抗原建立ELISA方法;KLH-FB1作为免疫原免疫8周龄BALB/C小鼠,应用细胞融合技术制备单克隆抗体。经三次亚克隆后,筛选出两株(6H3、6H11)能特异、稳定分泌抗FB1单克隆抗体的杂交瘤细胞株,并选择其中一株细胞通过体内诱生腹水的方法制备单克隆抗体。经检测表明单克隆抗体的亚型为IgG1,轻链为κ型。采用Protein G亲和层析法纯化腹水中的单克隆抗体后,用SDS-PAGE对其纯度进行鉴定,结果发现单克隆抗体的纯度高,活性较好。采用间接ELISA测得纯化后腹水效价高达l:16000。特异性检测实验证实该抗体与玉米赤霉烯酮、黄曲霉素B1等结构类似物几乎不存在交叉反应,与其它结构的兽药氯丙嗪、链霉素等无交叉反应。
利用获得的抗FB1单克隆抗体,分别建立了间接竞争ELISA检测方法、间接竞争磁酶免疫法与化学发光ELISA检测方法。
小分子的ELISA测定法一般有二种竞争检测模式:抗体包被直接竞争法、抗原包被直接竞争法以及间接竞争法。本研究利用制备的抗FB1单克隆抗体建立了抗原包被间接竞争ELISA法对FB1进行检测。间接竞争ELISA检测法,在包被抗原0.5μg/mL,一抗稀释度1:4000,二抗稀释度1:8000的条件下,检测灵敏度为0.44ng/mL,检测线性范围为0.44-73.06ng/mL,加标回收率在线性范围内达到78.4%-102%。虽然间接竞争ELISA方法比较繁琐,消耗的时间也相对长些,但是从结果看,与相关文献中的检测限对比,本实验的间接竞争ELISA检测灵敏度要高于直接竞争ELISA。并且,利用单克隆抗体建立的间接竞争ELISA方法,相比基于多克隆抗体建立的方法检测范围更广,灵敏度特异性更好。
磁酶免疫检测方法是免疫磁珠在免疫检测领域最主要的应用,其采用免疫磁珠配合常规ELISA方法进行检测,具有良好的检测效果。本研究以磁性微球作为检测的固相载体,首先通过制备兔抗KLH多克隆抗体,使其于磁珠结合后,再将KLH-FB1的偶联物包被与磁珠表面,形成外层包被有伏马毒素-KLH偶联物的免疫磁珠。结合间接竞争ELISA建立了磁酶免疫检测的方法。该方法在免疫磁珠稀释度1:20,一抗稀释度1:4000,二抗稀释度1:10000的条件下,检测灵敏度为0.24ng/mL,检测线性范围为0.54-26.3ng/mL,加标回收率在线性范围内达到80.4%-114.7%。由于免疫磁珠可均匀分布在样品溶液中,更易于接近靶物质,从而加快抗体与靶物质的结合速度,缩短了反应时间,从而提高了检测速率。此外,免疫磁珠可以利用磁性分离器方便地对所形成的复合物富集分离,使得洗涤结果更彻底、干扰物浓度极大降低和靶物质浓度有效富集,进而提高分析方法的信噪比和灵敏度。
化学发光ELISA法是将免疫反应和化学发光反应相结合以检测抗原或抗体的免疫技术,化学发光酶免疫分析属酶免疫分析,只是酶反应的底物是发光剂,其余操作步骤与酶免分析完全相同。化学发光ELISA检测在包被抗原0.5μg/mL,一抗稀释度1:4000,二抗稀释度1:30000的条件下,检测灵敏度为0.13ng/mL,检测线性范围为0.28-37.2ng/mL。加标回收率在线性范围内达到82.3%-108.3%。该方法的检测下限为0.13ng/mL,可用于检测FB1浓度较低的样品。与IMB-ELISA法相比,它具有更高的灵敏度,同时具有免疫分析法的成本低、速度快、特异性强、样品与处理简单和选择性强等优点,具有很大的发展前景。
将3种检测方法与市售商品化的试剂盒进行比对,检测敏感度均高于试剂盒,符合检测要求,适合于谷物类产品及动物饲料中FB1的安全监测和风险评估,均可进一步开发为检测试剂盒。
Fumonisins are a group of mycotoxins which are toxic and carcinogenicity mainly produced by Fusarium moniliforme. It usually happens in grain crops like maize and maize-based food and animal feed. The toxins can cause many diseases such as leukoencephalomalacia in horses, pulmonary edema in pigs and esophageal cancer in humans. It becomes a serious threat to human and animal, so more and more people pay attention on it. Among fumonisins, B1 is the most abundant and most toxic. In this study, an indirect competitive ELISA, Immunomagnctic beads ELISA and chemiluminecense ELISA were developed to analyze FB1 in food samples.
FB1 must be connected to a vector protein in order to gain immunogenicity because it's a small molecule which can not stimulate immune response. FB1 was conjugated using glutaraldhyde to ovalbumin(OVA) for use as a coating antigen for the ELISAs. According to the similar methods, FB1 was connected to keyhole limpet hemocyanin (KLH) for use as immunogen which was used to immunize 8-week-old BALB / c mouse to develop the monoclonal antibody. After three times of subcloning, 2 hybridoma cell lines stably secreted specific antibody against FB1 were screened by indirect competitive ELISA. The immunological sub-type IgG1κof the monoclonal antibody was determined. The antiserum was purified by proteinG chromatography respectively. The ascites monoclonal antibodies with titre l: 16000 were purified and characterized by SDS-PAGE. It showed that the purified antibody chromatography had higher purity and titer. AflatoxinB1, Chlorpromazine , streptomycin and Zearalenone showed no cross-activity with monoclonal antibodies.
Indirect competitive ELISA(IC-ELISA) , Immunomagnctic beads ELISA(IMB-ELISA) and chemiluminescence ELISA(CL-ELISA) were established respectively on the base of specific antibody against FB1.
ELISA is widely used detection method. There are two competition models in IC-ELISA: Direct antibody or antigen coated ELISA and indirect competition ELISA. IC-ELISA on condition that antigen coated for 4h under 37℃, concentration of coating antigen was 0.5μg/mL, anti-FB1 monoclonal antibody was diluted to 1:4000 and goat anti-rabbit IgG:HRP was diluted to 1:8000, the detection sensitivity of IC-ELISA was 0.44ng/mL and the detection range was 0.44-73.06ng/mL. Although IC-ELISA is time-consuming,the detection sensitivity and the detection range are better than others.
Using immunomagnetic beads(IMBs) as solid-phase carrier coated KLH-FB1. In the IMB-ELISA, the parameters were as follows: Immunomagnetic beads was diluted to 1:20, anti-FB1 antibody was diluted to 1:4000, the goat anti-rabbit IgG:HRP was diluted to 1:10000. The detection sensitivity was 0.24ng/mL and the detection range was 0.54-26.3ng/mL. The good enrichment and fast reaction rate increase the detection sensitivity.
In the CL-ELISA, the parameters were as follows: coating antigen was 0.5μg/mL, anti-FB1 antibody was diluted to 1:4000, the goat anti-rabbit IgG:HRP was diluted to 1:30000. The detection sensitivity was 0.13ng/mL and the detection range was 0.28-37.2ng/mL. This method has a good development prospect.
The detection sensitivity of the three methods was better than FB1 detection kit. The three methods and FB1 detection kit were used to detect FB1 in samples and recoveries were all over 80% in the detection range respectively.
FB1是一种不具备免疫原性的半抗原小分子,必须通过与载体蛋白的偶联获得免疫原性。本文通过戊二醛法将半抗原FB1与载体蛋白钥孔血蓝蛋白(KLH)和蛋白卵清白蛋白(OVA)分别偶联成为完全抗原KLH-FB1与OVA-FB1,OVA-FB1作为包被抗原建立ELISA方法;KLH-FB1作为免疫原免疫8周龄BALB/C小鼠,应用细胞融合技术制备单克隆抗体。经三次亚克隆后,筛选出两株(6H3、6H11)能特异、稳定分泌抗FB1单克隆抗体的杂交瘤细胞株,并选择其中一株细胞通过体内诱生腹水的方法制备单克隆抗体。经检测表明单克隆抗体的亚型为IgG1,轻链为κ型。采用Protein G亲和层析法纯化腹水中的单克隆抗体后,用SDS-PAGE对其纯度进行鉴定,结果发现单克隆抗体的纯度高,活性较好。采用间接ELISA测得纯化后腹水效价高达l:16000。特异性检测实验证实该抗体与玉米赤霉烯酮、黄曲霉素B1等结构类似物几乎不存在交叉反应,与其它结构的兽药氯丙嗪、链霉素等无交叉反应。
利用获得的抗FB1单克隆抗体,分别建立了间接竞争ELISA检测方法、间接竞争磁酶免疫法与化学发光ELISA检测方法。
小分子的ELISA测定法一般有二种竞争检测模式:抗体包被直接竞争法、抗原包被直接竞争法以及间接竞争法。本研究利用制备的抗FB1单克隆抗体建立了抗原包被间接竞争ELISA法对FB1进行检测。间接竞争ELISA检测法,在包被抗原0.5μg/mL,一抗稀释度1:4000,二抗稀释度1:8000的条件下,检测灵敏度为0.44ng/mL,检测线性范围为0.44-73.06ng/mL,加标回收率在线性范围内达到78.4%-102%。虽然间接竞争ELISA方法比较繁琐,消耗的时间也相对长些,但是从结果看,与相关文献中的检测限对比,本实验的间接竞争ELISA检测灵敏度要高于直接竞争ELISA。并且,利用单克隆抗体建立的间接竞争ELISA方法,相比基于多克隆抗体建立的方法检测范围更广,灵敏度特异性更好。
磁酶免疫检测方法是免疫磁珠在免疫检测领域最主要的应用,其采用免疫磁珠配合常规ELISA方法进行检测,具有良好的检测效果。本研究以磁性微球作为检测的固相载体,首先通过制备兔抗KLH多克隆抗体,使其于磁珠结合后,再将KLH-FB1的偶联物包被与磁珠表面,形成外层包被有伏马毒素-KLH偶联物的免疫磁珠。结合间接竞争ELISA建立了磁酶免疫检测的方法。该方法在免疫磁珠稀释度1:20,一抗稀释度1:4000,二抗稀释度1:10000的条件下,检测灵敏度为0.24ng/mL,检测线性范围为0.54-26.3ng/mL,加标回收率在线性范围内达到80.4%-114.7%。由于免疫磁珠可均匀分布在样品溶液中,更易于接近靶物质,从而加快抗体与靶物质的结合速度,缩短了反应时间,从而提高了检测速率。此外,免疫磁珠可以利用磁性分离器方便地对所形成的复合物富集分离,使得洗涤结果更彻底、干扰物浓度极大降低和靶物质浓度有效富集,进而提高分析方法的信噪比和灵敏度。
化学发光ELISA法是将免疫反应和化学发光反应相结合以检测抗原或抗体的免疫技术,化学发光酶免疫分析属酶免疫分析,只是酶反应的底物是发光剂,其余操作步骤与酶免分析完全相同。化学发光ELISA检测在包被抗原0.5μg/mL,一抗稀释度1:4000,二抗稀释度1:30000的条件下,检测灵敏度为0.13ng/mL,检测线性范围为0.28-37.2ng/mL。加标回收率在线性范围内达到82.3%-108.3%。该方法的检测下限为0.13ng/mL,可用于检测FB1浓度较低的样品。与IMB-ELISA法相比,它具有更高的灵敏度,同时具有免疫分析法的成本低、速度快、特异性强、样品与处理简单和选择性强等优点,具有很大的发展前景。
将3种检测方法与市售商品化的试剂盒进行比对,检测敏感度均高于试剂盒,符合检测要求,适合于谷物类产品及动物饲料中FB1的安全监测和风险评估,均可进一步开发为检测试剂盒。
Fumonisins are a group of mycotoxins which are toxic and carcinogenicity mainly produced by Fusarium moniliforme. It usually happens in grain crops like maize and maize-based food and animal feed. The toxins can cause many diseases such as leukoencephalomalacia in horses, pulmonary edema in pigs and esophageal cancer in humans. It becomes a serious threat to human and animal, so more and more people pay attention on it. Among fumonisins, B1 is the most abundant and most toxic. In this study, an indirect competitive ELISA, Immunomagnctic beads ELISA and chemiluminecense ELISA were developed to analyze FB1 in food samples.
FB1 must be connected to a vector protein in order to gain immunogenicity because it's a small molecule which can not stimulate immune response. FB1 was conjugated using glutaraldhyde to ovalbumin(OVA) for use as a coating antigen for the ELISAs. According to the similar methods, FB1 was connected to keyhole limpet hemocyanin (KLH) for use as immunogen which was used to immunize 8-week-old BALB / c mouse to develop the monoclonal antibody. After three times of subcloning, 2 hybridoma cell lines stably secreted specific antibody against FB1 were screened by indirect competitive ELISA. The immunological sub-type IgG1κof the monoclonal antibody was determined. The antiserum was purified by proteinG chromatography respectively. The ascites monoclonal antibodies with titre l: 16000 were purified and characterized by SDS-PAGE. It showed that the purified antibody chromatography had higher purity and titer. AflatoxinB1, Chlorpromazine , streptomycin and Zearalenone showed no cross-activity with monoclonal antibodies.
Indirect competitive ELISA(IC-ELISA) , Immunomagnctic beads ELISA(IMB-ELISA) and chemiluminescence ELISA(CL-ELISA) were established respectively on the base of specific antibody against FB1.
ELISA is widely used detection method. There are two competition models in IC-ELISA: Direct antibody or antigen coated ELISA and indirect competition ELISA. IC-ELISA on condition that antigen coated for 4h under 37℃, concentration of coating antigen was 0.5μg/mL, anti-FB1 monoclonal antibody was diluted to 1:4000 and goat anti-rabbit IgG:HRP was diluted to 1:8000, the detection sensitivity of IC-ELISA was 0.44ng/mL and the detection range was 0.44-73.06ng/mL. Although IC-ELISA is time-consuming,the detection sensitivity and the detection range are better than others.
Using immunomagnetic beads(IMBs) as solid-phase carrier coated KLH-FB1. In the IMB-ELISA, the parameters were as follows: Immunomagnetic beads was diluted to 1:20, anti-FB1 antibody was diluted to 1:4000, the goat anti-rabbit IgG:HRP was diluted to 1:10000. The detection sensitivity was 0.24ng/mL and the detection range was 0.54-26.3ng/mL. The good enrichment and fast reaction rate increase the detection sensitivity.
In the CL-ELISA, the parameters were as follows: coating antigen was 0.5μg/mL, anti-FB1 antibody was diluted to 1:4000, the goat anti-rabbit IgG:HRP was diluted to 1:30000. The detection sensitivity was 0.13ng/mL and the detection range was 0.28-37.2ng/mL. This method has a good development prospect.
The detection sensitivity of the three methods was better than FB1 detection kit. The three methods and FB1 detection kit were used to detect FB1 in samples and recoveries were all over 80% in the detection range respectively.
引文
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[33]丛宪玲,张德宝,吴秀丽等.采用二氧化硅微球法回收DNA的实验研究中国免疫学杂志. 2004. 20:341-343.
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[42]贾秀鹏,张东生,郑杰等.用于肿瘤热疗的锰锌铁氧体纳米粒了的制备及表征.生物医学工程学杂志. 2006, 23:1263.
[43] Singer, J.M, C.M. Plotz. The Latex Fixation Test: Application to the serologic diagnosis of rheumatoid arthritis. Am J Med. 1956, 21: 888.
[44] Yanyan Wang, Hong Xu, Meng Wei. Study of superparamagnetic nanoparticles as labels in quantitative lateral flow immunoassay. Materials Science and Engineering C 29. 2009:714-718.
[45]曹馨匀,张云涛,谢雍等.利福霉素单克隆抗体的制备及竞争ELISA检测方法的建立.中国生物制品杂志. 2009, 22(3):272-275.
[46]宫慧芝,计融,杨军等.伏马毒素B1免疫学检测方法的建立[J].中国公共卫生. 2006, 22(7): 840-842.
[47]王硕,王向红,郑文杰等.采用酶联免疫检测技术定量检测食品中伏马毒素B1含量的试剂盒及其检测方法[P].中国专利: CN200710056559.6, 2008-07-30.
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[50]王君.伏马毒素B1免疫学检测方法的研究[D].中国优秀硕士学位论文全文数据库.2010.
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[26] Teresa Gazzotti, Barbara Lugoboni, Elisa Zironi et al. Determination of fumonisin B1 in bovine milk by LC–MS/MS[J]. Food Control. 2009, 20:1171-1174
[27] Marilena Muscarella, Sonia Lo Magro, Donatella Nardiello et al. Development of a new analytical method for the determination of fumonisins B1 and B2 in food products based on high performance liquid chromatography and fluorimetric detection with post-column derivatization[J]. Jourmal of Chromatography A. 2008, 1203:88-93.
[28] Ying Quan, Yan Zhang, Shuo Wang et al. A rapid and sensitive chemiluminescence enzyme-linked immunosorbent assay for the determination of fumonisin B1 in food samples[J]. Analytica Chimica Acta. 2006,580:1-8.
[29]李美佳.当代免疫学技术与应用.北京:北京医科大学国协和医科大学联合出版社. 1998, 549～561.
[30] Karl Holschuh, Achim Schwammle. Preparative purification of antibodies with protein A an alternative to conventional chromatography. Journal of Magnetism and Magnetic Materials. 2005, 293:345–348
[31]Ivo Safarika, Mirka Safarkova. Use of magnetic techniques for the isolation of cells. Journal of Chromatography B. 1999, 722:33–53.
[32]张立明.磁性微球的制备及其在基因组脱氧核糖核酸提取上的应用[硕士论文].天津大学. 2005.
[33]丛宪玲,张德宝,吴秀丽等.采用二氧化硅微球法回收DNA的实验研究中国免疫学杂志. 2004. 20:341-343.
[34]Kharkevich D A, Alyautdin R N, Filippov V I. Employment of magnet-susceptible microparticles for the targeting of drugs. Journal of Pharmacy and Pharmacology. 1989, 41 286~288.
[35]Gupta P K, Hung C T. Albumin microspheres.V.Evaluation of parameters controlling the efficacy of magnetic microspheres in the targeted delivery of adriamycin in rats International Journal of Pharmaceutics. 1990, 59:57~67.
[36]Liang Z, Lauterbur P.C.. Principles of Magnetic Resonance Imaging: A Signal ProcessingPerspective. in IEEE Press Series in Biomedical Engineering. 2000, 107-136.
[37]马隽.磁共振成像在甲状旁腺腺瘤诊断中的应用.中国地方病防治杂志. 2009, 24(5):397-398.
[38]张旭良,张影.肿瘤热疗中的儿个医学工程问题医疗设备信息. 2002, 3:1.
[39]王煦漫,张彩宁.纳米材料在肿瘤磁热疗中的应用及展望纳米材料与应用. 2007, 4(3):15-17.
[40]Gilchrist K, Medal R, Shorey D. Selective Inductive Heating of Lymph Nodes[J]. Annals of Surgery. 1957, 146:596.
[41]Sonvico F, Mornet S.Vasseur S, et al. Folate-conjugated Iron Oxide Nanoparticles for Solid Tumor Targeting as Potential Specific Magnetic Hyperthermia Mediators: Synthesis. Physicochemical Characterization, and in Vitro Experiments Bioconjugate Chemistry. 2005, 16:1181.
[42]贾秀鹏,张东生,郑杰等.用于肿瘤热疗的锰锌铁氧体纳米粒了的制备及表征.生物医学工程学杂志. 2006, 23:1263.
[43] Singer, J.M, C.M. Plotz. The Latex Fixation Test: Application to the serologic diagnosis of rheumatoid arthritis. Am J Med. 1956, 21: 888.
[44] Yanyan Wang, Hong Xu, Meng Wei. Study of superparamagnetic nanoparticles as labels in quantitative lateral flow immunoassay. Materials Science and Engineering C 29. 2009:714-718.
[45]曹馨匀,张云涛,谢雍等.利福霉素单克隆抗体的制备及竞争ELISA检测方法的建立.中国生物制品杂志. 2009, 22(3):272-275.
[46]宫慧芝,计融,杨军等.伏马毒素B1免疫学检测方法的建立[J].中国公共卫生. 2006, 22(7): 840-842.
[47]王硕,王向红,郑文杰等.采用酶联免疫检测技术定量检测食品中伏马毒素B1含量的试剂盒及其检测方法[P].中国专利: CN200710056559.6, 2008-07-30.
[48] Ildiko B.V, Erzse S, Be′la F, et al. Development of a Sensitive ELISA for the Determination of Fumonisin B1 in Cereals.J. Agric. Food Chem. 2000, 48:2821-2825.
[49] Hanne R. C, Feng Y.Y, Fun S. C. Development of a Polyclonal Antibody-Based Sensitive Enzyme-Linked Immunosorbent Assay for Fumonisin B4.J. Agric. Food Chem. 2000, 48:1977-1984.
[50]王君.伏马毒素B1免疫学检测方法的研究[D].中国优秀硕士学位论文全文数据库.2010.
[51] Gosling J P. 1mmunoassays A Practical Approach. America: Oxford University Press. 2000: 41.
[52]杨利国,胡少旭,魏平华.酶免疫测定技术.南京:南京大学出版社. 1998: 13.
[53]林学颜,张玲.现代细胞与分子免疫学.北京:科学出版社. 1999: 459-478.
[54]常超.动物可食性组织中呋喃唑酮残留标示物ELISA检测方法及试剂盒研制[D].武汉:华中农业大学. 2007.
[55]王海涛,魏慧娟,马吉林等.食管癌高发区玉米中伏马菌素B1的检测[J].肿瘤防治研究. 1999, 26(3):168-170.
[56] SN /T 1958-2007.进口食品中伏马毒素B1残留量检测方法[S].
[57] Shetll H B, Sporns P. DeveIopment of asingle ELISA for detection of Sulfanamides[J]. Agric Food Chem. 1991, 39(9):1696-1700.
[58]郭云昌,刘秀梅,刘江.伏马菌索s,免疫检测方法的研究[J].卫生研究. 1999, 28(4): 238- 240.
[59] Nishizono I, lida S.Suzuki N, et al. Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers[J]. Clin Chem. 1991, 37(9):1639-1644.
[60]张文艳.增强辣根过氧化物酶催化鲁米诺化学发光反应的新型增强剂四苯硼钠[J].分析化学. 1998, 26(11):100-102.
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