【作者】 姜金庆；

【导师】 王建华；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2011， 博士

【摘要】 19–去甲睾酮(NT)用于治疗骨质疏松症,蛋白质缺陷型疾病和烧伤等;它还可以提高运动员和赛马比赛的成绩,增强食源性动物瘦肉生产率。但NT及代谢物残留可引起人的肝、脑功能障碍、心血管系统受损和内分泌失调等副作用,因此在世界上多数国家,除医疗和科研之外,禁止使用。NT残留检测的传统方法有气相色谱–质谱法(GC–MS),液相色谱质谱联用法(LC–MS)及其它定量检测方法。这些方法需要专业的技术人员、繁琐的样品前处理过程和昂贵的仪器设备,不适合大批量市场检测和现场监控。而免疫学检测技术灵敏高、特异性强、操作简单、价格低廉,能够实现对生物液体的小体积、大通量检测,逐渐成为有毒有害残留物检测的替代方法之一。本文对NT人工抗原的合成、多克隆抗体(pAb)和单克隆抗体(mAb)的制备、间接竞争ELISA和直接竞争ELISA检测方法的优化、试剂盒检测性能及应用、免疫金标试纸条的研制和气质联用(GC–MS)确证法等进行了研究,主要结果如下:1.丁二酸酐法衍生化NT,制备17β–19–NT琥珀酸酯半抗原,质谱和核磁共振1H–NMR谱表明合成成功。分别用EDC法和混合酸酐法将半抗原与BSA和OVA偶联,制备免疫抗原和检测抗原,并进行紫外扫描和红外光谱鉴定,NT与BSA偶联比为18:1。NT–BSA免疫Balb/C小鼠的抗血清效价达到1∶25600,间接竞争ELISA的IC50为27.3 ng/mL。纯化后的抗血清与α–NT的交叉反应率为69%,与其它激素的交叉反应率均<0.05%。结果表明:人工抗原合成成功,为试剂盒和试纸条的研制提供基础材料。2. NT–17–BSA免疫原制备兔抗多克隆抗体,用NT–3–OVA检测抗原包板,建立异源性间接竞争ELISA试剂盒。对NT的线性检测范围为0.06 ~ 31.2 ng/mL,半数抑制浓度(IC50)和最低检测限(LOD)分别为1.55和0.04 ng/mL。检测缓冲液中pH值和磷酸根离子浓度的最佳参数分别为7.4 mM和10 mM;对甲醇和NaOH的最高容忍度分别为30%和10%。多克隆抗体对群勃龙和雌二醇的交叉反应率分别为9.6%和6.4%;猪尿、牛尿、狗尿和马尿简单稀释1∶20、1∶20、1∶20和1∶50可有效消除基质效应性;动物空白尿液基质中的添加回收率为85% ~ 110%,相对标准差为4.4% ~ 16.3%。试剂盒4℃下保存期至少6个月,温育过程优化后可节约检测时间30 ~ 90 min。3.细胞融合技术筛选出5株NT mAb杂交瘤细胞株,命名为NT–1、NT–2、NT–3、NT–4和NT–5。细胞株单抗腹水效价为0.64 ~ 2.56×105,IC50为0.55 ~ 1.0 ng/mL;抗体为IgG1亚型,具k轻链;亲和常数(Ka)为2.6 ~ 4.7×109 L/mol。建立的icELISA标准曲线在PBS中的的线性检测范围为0.004 ~ 85.8 ng/mL,IC50值为0.55 ng/mL,检测限(LOD)为0.002 ng/mL;抗体与α–NT的交叉反应率(CR)为62%,与其它化合物无CR。ELISA方法和液质联用检测(LC–MS/MS)的相关性良好(R2 = 0.9871)。4.细胞融合技术筛选出3B8–E6单克隆杂瘤细胞株,建立异源性直接竞争ELISA(dcELISA)检测方法,其线性检测范围为0.004 ~ 19 ng/mL,IC50值为0.28 ng/mL,检测限(LOD)为0.002 ng/mL。抗体与勃地酮和群勃龙的交叉反应率分别为6.9%和1.2%,与其它化合物无CR。标准曲线对甲醇有较高的容忍度(30%),而检测缓冲液中吐温–20的最佳含量为0.05%,乙腈和丙酮不能超过10%。4℃和–20℃保存条件下,试剂盒有效期分别为180 d和240 d。简单稀释法进行样品前处理可以消除基质效应,ELISA试剂盒和GC–MS检测的相关系数(R2)分别为0.9918(牛肉)、0.9834(牛肝)和0.9976(牛肾)。5.研制NT残留检测的胶体金免疫层析试纸条。柠檬酸钠还原法制备直径15 nm的胶体金颗粒,pH 9.0条件下制备金标单克隆抗体复合物,竞争法原理组装检测试纸条。试纸条目测灵敏度为1.6 ng/mL,实际检测线确定为8 ng/mL,与群勃龙和勃地酮的交叉反应率(CR)分别为15.6%和7.8%,与其它化合物无CR。试纸条在4℃和室温条件下,有效期分别为12个月和6个月。6.建立NT残留检测的GC–MS检测方法。动物组织在醋酸缓冲液中酶解,用甲醇提取,正已烷去脂,提取液经SPE–C18柱净化,使用七氟丁酸酐衍生化。电子轰击离子源(EI),不分流进样,阳离子电离(PCI),质谱图四个定量离子为m/z 666、453、318和306。在1 ~ 20 ng/g范围内,线性回归方程式为Y=– 68 354 + 467 084X (R2 = 0.9997)。动物组织中添加回收实验的回收率为63% ~ 101%,变异系数(CV)为2.7% ~ 8.9%。更多还原

【Abstract】 19–Nortestosterone (NT) is often used in veterinary as well as in human medicine for the treatment of protein deficiency diseases and osteoporosis. This anabolic androgenic steroid has also been employed as a doping agent to boost muscular strength and performance in sports and horse racing, and as a growth–promoting agent to accelerate weight gain and improve feeding efficiency in animals. But NT and it metabolite residues damage liver functions and cardiovascular system, cause endocrine dyscrasia, cerebral dysfunction, emotional instability and other side–effects. Therefore, it is forbidden in most countries, except for medical treatment and scientific research.Traditionally, NT residue analysis has relied upon gas chromatography coupled to mass spectrometry (GC–MS) or liquid chromatography coupled to mass spectrometry (LC–MS), and other classical analytical methods. Chromatographic techniques provide sensitive and confirmatory analysis, however, they usually require highly skilled personnel and expensive equipments. Moreover, these laborious sample pretreatment procedures involve numerous extraction steps that are time–consuming and unsuitable for routine analysis of a large number of samples or on–site determinations. Compared with instrumental methods, immunoassays are portability and cost–effective, with adequate sensitivity, high selectivity, and simple sample extraction process. Therefore, immunochemical techniques become a popular and are increasingly considered as alternative or complementary methods for residue analysis nowadays.The objective of this study was to develop a rapid immunoassay for detection of NT residue in animal–origin products, including the synthesis and identification of artificial antigen, preparation and characterization of NT polyclonal antibody and monoclonal antibodies, NT–kits development and optimization, the preparation of immunological gold–labled strip, and the development of GC–MS analysis.1. Succinic anhydride method was employed to prepare NT–17–(succinic anhydride) ester, which was proved by (+)ESI–MS spectrum and 1H–NMR spectrogram. The 17β–19–NT hapten was conjugated to BSA as immunogen with the EDC method, and to OVA as detection antigen by mixed–anhydride technology respectively. The results of infrared spectra (IR) and UV–visible spectra indicated that the artificial antigen was synthesized successfully and the conjugation ratio of NT–BSA was 18:1. Balb/C mice immunized with NT–BSA were introduced to obtain NT pAb, whose antibody titer for indirect ELISA was 1:25600, and IC50 value was 27.3 ng/mL by icELISA. The antisera cross–reacted withα–NT about 69%, but not higher than 0.05% to other steroids tested. The results showed that the artificial antigen was synthesized successfully, which lay a solid foundation for preparing NT–Kits and a rapid test strip.2. An indirect heterologous competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody for analysis of NT in animal urines was developed. This assay was sensitive and had a linear range from 0.06 to 31.2 ng/mL, with IC50 and LOD values of 1.55 ng/mL and 0.04 ng/mL, respectively. After optimization, the best parameters for pH value and phosphate ion concentration were determined to be 7.4 and 10 mM in assay buffer. It also indicated that the concentrations of methanol and NaOH in dilute solution should not be higher than 30% and 10%. Except for cross–reactivity (CR) toward trenbolone (9.6%) and estradiol (6.4%), no significant CR was observed for other analogues tested. To reduce the matrix interference, 1:20, 1:20, 1:20 and 1:50 dilution folds were chosen for pork, beef, dog and horse urine, respectively. The recoveries of NT spiked in the four matrix were in the range of 85 ~ 110%, with RSD values from 4.4% to 16.3%. when stored at 4℃, the NT–Kits could preserve 6 months or longer, and the program could save 30 ~ 90 min after the incubation process was optimized.3. Five hybridoma cell lines named NT–1, NT–2, NT–3, NT–4 and NT–5 were screened out through cell fusion technology, and the corresponding mAbs were of the IgG1 isotype with a k light chain, and the affinity constants (Ka) of all mAbs were between 2.6 and 4.7×109 L/mol. The titer and IC50 values of purified ascites were in the range of 0.64 ~ 2.56×105 and 0.55 ~ 1.0 ng/mL respectively. Based on the NT–1 hybridoma, a heterologous icELISA method was developed for the quantitative detection of NT, which dynamic range was from 0.004 to 85.8 ng/mL, with a LOD and IC50 values of 0.002 ng/mL and 0.55 ng/mL, respectively. Except for a high cross-reactivity (62%) toα–NT, negligible cross–reactivity to other compounds was observed. The correlation coefficient between the established icELISA and LC–MS/MS method was excellent (R2 = 0.9871).4. Based on the hybridoma of 3B8–E6, an heterologous direct competitive ELISA (dcELISA) method to detect NT residue was developed. Under the optimal conditions, this assay exhibited an working range of 0.004 ~ 19 ng/mL, with IC50 and LOD values of 0.28 and 0.002 ng/mL, respectively. Except for a minor cross–reactivity withβ–boldenone (6.9%) and trenbolone (1.2%), the other interference to this assay was negligible (<0.05%). The dcELISA can tolerate higher concentrations of methanol (30%), while the best contents of Tween–20 was determined to be 0.05%, and acetonitrile and acetone should not be higher than 10% in assay buffer. The stabilization studies showed that the dcELISA kits can be stored for at least 180 and 240 days, at 4℃a nd–20℃, respectively. After three sample pretreatment procedures checked, simple dilution method was recommended for further use. When applied to bovine samples, the correlation coefficients (R2) of ELISA and GC–MS data were 0.9918 in muscle, 0.9834 in liver, and 0.9976 in kidney.5. This chapter presents the generation of colloidal gold–based strip to detect NT residue in animal tissues. Colloidal gold particles with the diameter of 15 nm were prepared by the trisodium citrate reduction method, colloidal gold labelled NT mAb as a detection agent was conjugated under pH 9.0, and the immunochromatographic strip was assembled according to the competition theory. The visual sensitivity of strip was 1.6 ng/mL, therefore the detection limit was determined to be 8 ng/mL after 5–fold sample dilution. Of all the analogues, this assay exhibited cross–reactivities to trenbolone (15.6%) andβ–boldenone (7.8%), but negligible CR values (<0.05%) to other chemicals tested. Under 4℃and room temperature, the strip can be validitied for at least 12 and 6 months, respectively.6. A GC–MS method for determination of NT residues has been developed. Animal tissues were hydrolyzed in an acetate buffer, and the homogenate was extracted with methanol, then the fat was deprived with n–hexane. The analytes were subjected to a SPE C18 cartridge for clean–up, and the dried extracts were derivatized with heptafluorobutyric anhydride (HFBA). Using electron impact mass spectrometry (EI–MS) with positive chemical ionization (PCI), four diagnostic ions (m/z 666, 453, 318 and 306) were determined. An equation of linear regression with Y=– 68 354 + 467 084X (R2 = 0.9997) was obtained, over the concentration of 1 ~ 20 ng/g for NT. When applied to spiked samples in animals, the recoveries ranged from 63% to 101%, with CV values of 2.7% ~ 8.9%.更多还原