【作者】 徐晓丽；

【导师】 战文斌； 绳秀珍；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2011， 博士

【摘要】 病毒性疾病严重威胁着水产养殖业的健康快速发展。鉴于目前病毒病尚无有效的治疗方法,病毒的早期准确检测对疾病的预防和控制尤为重要,因此建立一种多样品、多指标检验并行处理的简便、快速、灵敏、准确的病毒检测方法对病毒病的防控具有重要意义。新兴的抗体芯片技术结合了抗原抗体反应的特异性和芯片高密度集成优势,只需少量生物样品,一次检测便可获得几种甚至几万种有关的生物信息或疾病的检测结果,在人类疾病中病原/疾病标志物等的检测上已显示出广阔的应用前景。但尚未发现有利用抗体芯片法检测水产动物病毒的相关报道。本文将单克隆抗体(单抗)技术、免疫标记技术和抗体芯片技术相结合,以目前水产养殖中的重要病毒病的病原对虾白斑症病毒(White Spot Syndrome Virus, WSSV)和鱼类淋巴囊肿病毒(Lymphocystis Disease Virus, LCDV)为模式病毒,建立了水产动物病毒检测抗体芯片制备体系,优选了芯片载体,优化了反应条件,研究了点样缓冲液、抗体浓度、固定时间、封闭剂种类和浓度、抗原-抗体反应时间、不同标记显色方法等对芯片灵敏度的影响,制备了对虾WSSV检测抗体芯片和鱼类LCDV检测抗体芯片,并将其用于WSSV/LCDV的检测。具体研究结果如下:1.针对芯片载体的选择和处理是制备高质量抗体芯片的关键,本文制备了琼脂糖凝胶、丙烯酰胺凝胶、APES、醛基、巯基、多聚赖氨酸修饰玻片,综合分析了以上6种不同修饰的玻片及氨基化玻片、硝酸纤维素(NC)膜、PVDF膜共9种不同载体对抗体的固定效率和效果。结果显示1.2%琼脂糖凝胶修饰玻片表面信号点圆润、均匀,无边缘扩散和拖尾现象,信号值最高,而其他载体都有不同程度的拖尾现象,表明琼脂糖凝胶修饰玻片对抗体的固定能力和固定效果最好。原子力显微镜显示琼脂糖凝胶修饰玻片表面为均匀的三维多孔结构,平均表面粗糙度为18.6 nm,结合琼脂糖凝胶表面经NaIO4活化后的醛基基团,能够以物理吸附和共价结合的方式牢固的固定抗体。因此后续实验采取琼脂糖凝胶修饰玻片作为抗体芯片的载体。2.以WSSV/LCDV病毒粒子为模式病毒,采用双抗体夹心方式建立了水产动物病毒的抗体芯片检测体系。提纯WSSV/LCDV病毒粒子,制备病毒的兔抗血清,纯化后进行特性分析。结果显示纯化后得到了高活性、高效价的兔抗WSSV及兔抗LCDV抗体,效价分别为1:64000和1:32000。复苏本研究室前期制备的WSSV/LCDV单抗杂交瘤细胞株,采用腹水生产方法获得大量WSSV/LCDV单抗,辛酸硫酸铵法结合Protein G亲和层析纯化后进行特性分析。结果显示纯化后WSSV/LCDV单抗效价均为1:32000,具很好的活性。采用Cy3抗体标记试剂盒(GE)对纯化后的高效价单抗作Cy3标记,制备了特异性检测WSSV/LCDV的抗体探针,Cy3标记后WSSV/LCDV单抗探针的工作浓度分别为1:2400和1:3000。3.以制备的兔抗WSSV/LCDV多克隆抗体为捕获抗体,用小型手动芯片点样系统将其点样于优选的琼脂糖凝胶修饰的芯片载体上,制备WSSV/LCDV检测抗体芯片。选用特异性强的Cy3标记的WSSV/LCDV单抗为检测抗体。抗体芯片与WSSV/LCDV病毒稀释液孵育形成复合物,该复合物被Cy3标记的特异性WSSV/LCDV单抗探针识别,经CCD芯片扫描仪读取结果。通过改变芯片制备及应用过程中的具体条件参数,对点样缓冲液、点样抗体浓度、固定时间、封闭剂种类和浓度、洗涤方法、抗原-抗体反应时间、不同标记显色方法进行了优化,得到芯片制备与应用的最佳条件。结果显示,点样用捕获抗体采用含50%甘油的PBS调整到合适浓度(兔抗WSSV抗体为0.1 mg/ml,兔抗LCDV抗体为0.5 mg/ml)进行点样,点样后芯片于37℃饱和湿度固定2 h,3%牛血清白蛋白于37℃饱和湿度封闭1 h,依次用dH2O、PBST、PBST洗涤,每次5 min,甩干后低温密封保存。芯片与捕获抗体的反应时间为15~30 min,检测抗体与芯片上的抗原抗体复合物的反应时间15~30 min,不要超过45 min。4.研究了不同载体、不同标记物(包括辣根过氧化物酶(HRP),Cy3,异硫氰酸荧光素(FITC),胶体金和生物素)标记单抗用作检测抗体、芯片保存时间等对检测灵敏度的影响。结果显示,以琼脂糖修饰玻片作为载体制备的抗体芯片,分别采用HRP和Cy3标记抗体探针作为检测抗体,检测灵敏度高,WSSV检测抗体芯片的灵敏度分别为0.15μg/ml和0.31μg/ml,LCDV检测抗体芯片的灵敏度均为0.55μg/ml。WSSV浓度在0.62μg/ml-9.9μg/ml范围内,荧光信号强度与病毒浓度的对数值与呈线性关系,相关系数为0.98925; LCDV浓度在0.55-17.56μg/ml范围内,信号强度与病毒浓度的对数值与呈线性关系,相关系数为0.9900。检测信号经生物素-链酶亲和素系统放大之后,灵敏度明显提高,WSSV检测抗体芯片可达25 ng/ml,LCDV检测抗体芯片可达70 ng/ml。Cy3标记抗体作为检测抗体,抗体芯片于-20℃密封保存4个月后,检测的背景值大幅升高,从而导致相对信号值的下降。HRP标记抗体作为检测抗体,芯片于-20℃下密封保存12个月,WSSV检测抗体芯片的检测灵敏度不变;LCDV检测抗体芯片灵敏度下降至1.1μg/ml。5.利用建立的抗体芯片技术平台,制备了WSSV检测抗体芯片和LCDV检测抗体芯片。将抗体芯片用于对虾WSSV和鱼类LCDV样品的检测,检测结果与酶联免疫吸附实验(Enzyme Linked Immunosor-bent Assay, ELISA)比较,结果显示WSSV检测抗体芯片与ELISA检测的结果符合率为100%,相关系数为0.9853;LCDV检测抗体芯片与ELISA检测的结果符合率为100%,相关系数为0.9802。说明所制备的抗体芯片能够准确的检测病原,具有很好的特异性与准确性。6.将抗体芯片技术与免疫酶技术结合,采用HRP标记单抗探针作为检测抗体,制备的WSSV/LCDV检测抗体芯片用于对虾WSSV与鱼类LCDV的现场检测,肉眼即可观察检测结果,解决了抗体芯片结果的读取依赖专用仪器的问题。可视化WSSV/LCDV检测抗体芯片用于病毒现场检测,操作简便,无需昂贵仪器,准确性与IIFA法和ELISA法一致,信号值与病毒浓度的对数值呈线性关系,相关系数分别为0.9505和0.9567,可在一定范围内对病毒进行相对定量检测,在苗种选育、养殖生产的疾病监测中有广阔的应用前景。本研究所建立的抗体芯片制备体系能够拓展到其它水产动物病原检测领域,是对传统免疫学检测手段的发展。构建的对虾WSSV检测抗体芯片和鱼类LCDV检测抗体芯片,能够对病毒进行准确检测,具微量化、特异性强、方法敏感、样品处理简单和实验条件易于控制等优点,可用于养殖动物WSSV/LCDV的实验室/养殖现场、进出口检疫中的多样品平行检测,具有广阔的应用前景,并为水产动物病原的多样品多病原平行检测提供了有效的技术参考。更多还原

【Abstract】 Virus is the most important lethal pathogen that obstructs the development of current aquaculture severely. As there is no effective drug for treatment of viral diseases, detecting virus pathogen at early stage in high efficiency and veracity is no doubt conducive to industry. Hence a novel method for parallel detection of multiple samples or pathogens in a convenient and simple way is urgently needed. Antibody-based microarray, combining the specificity of antigen-antibody reaction and high-density integration of microarray, is a novel proteomic technology that can meet the requirements which is a powerful tool for parallel detection of multiple parameters. It has the advantages of strong specificity, high sensitivity, simple sample handling, and high through-put analysis with minimal sample consumption. The antibody microarray has shown vast prospect on the detection of virus or biomarkers related to cancers in medicine. However, to our best knowledge, no research on protein microarray for pathogen detection of aquatic animals has been reported. In this paper, we developed the antibody microarray system by combining the monoclonal antibody technology, immune markers and antibody microarray technology. Taking white spot syndrome virus (WSSV) and lymphocystis disease virus (LCDV) as model virus, we prepared WSSV/LCDV antibody microarray and optimized the procedure conditions at multiple samples for microarray application. Also we investigated the sensitivity, specificity, accuracy of WSSV/LCDV antibody microarray and utilized the microarrays to detect WSSV/LCDV. Details are as follows.1. Nine kinds of supports (Poly-L-lysine, MPTS, aldehyde, APES, amine, polyacrylamide gel and agarose gel modified slides, Nitrocellulose membrane, PVDF membrane) were compared in the efficiency of immobilizing proteins. The results showed that spots on 1.2% agarose gel-modified slides were round and homogeneous without diffusion tailing on the verge, showing superior size and clear pattern, providing the highest signal value. These characters indicated the highest capacity of protein immobilization. The slides were characterized by atomic force microscope (AFM) and the surface of 1.2% agarose gel-modified slides displayed 3-dimensional structure with many holes and great thickness. Measurement with Nanoscope 5.12 software revealed that the mean roughness of agarose gel was 18.6 nm, which was much greater than other slides (1.45-4.9 nm). These results indicated that rougher mesoporous agarose gel surfaces with aldehyde functions might have higher latent capacity of protein adsorbing and covalent linking in their natural states. Taken together, slides modified with 1.2% agarose gel were chosen as appropriate supports for microarrays.2. Sandwich immunoassay was adopted to develop the antibody microarray for a higher sensitivity and specification. Purified WSSV/LCDV particles were used to immunize New Zealand white rabbits and rabbit anti-WSSV/LCDV antibodies were obtained. After purification we got polyclonal antibodies with high activity and titer. The titers of rabbit anti-WSSV antibody and anti-LCDV antibody were 1:64000 and 1:32000, respectively.Four anti-WSSV monoclonal antibodies (MAbs) (2E6, 2A3, 4G9 and 2D11) and four anti-LCDV MAbs (3G3, 2B6, 1D7 and 2D11), developed previously in our laboratory, were produced in ascites by injection the hybridoma clone into the peritoneal cavity of Balb/c mice individually, and purified with the Ampure PA kit as per the manufacturer’s protocol. Then these MAbs were mixed with equal proportion respectively and labeled with Cy3 according to the manufacturers’instructions. Cy3-conjugated anti-WSSV MAbs or anti-LCDV MAbs was 2400-fold or 3000-fold diluted and used as detection antibody.3. Rabbit anti-WSSV or anti-LCDV polyclonal antibody was diluted and arrayed as capture antibody of the microarray on the agarose gel-modified slides. After immobilization and blocking, the microarray slides were incubated with virus diluents and the antibody-antigen complex was detected by specific Cy3-conjugated anti-WSSV or anti-LCDV MAbs. The results were measured by a laser chipscanner and analyzed with Lab-chipscanner 2.0. To obtain satisfied fluorescence signal intensity, optimal conditions in printing buffer, capture antibody concentration, immobilization, blocking, washing, incubating time and markers were searched. The results illustrated that the optimum conditions were as follows. Rabbit anti-WSSV antibody was diluted to 0.1 mg/ml and rabbit anti-WSSV antibody was 0.5 mg/ml with PBS containing 50% glycerol as printing buffer. After arraying, the microarrays were put in a humid chamber at 37°C for 2 h to immobilize the antibody and then blocked by 3% BSA at 37°C for 1 h. The antibody microarray was washed by rinsing with dH2O, PBST, PBST in sequence for 5 min each and dried by centrifuging, then sealed at low temperature. Incubation was performed for 15~30 min at 37°C saturated humidity, and incubation longer than 45 min would cause a higher background value.4. We investigated influence of the supports, markers (Cy3, Horseradish peroxidase, FITC, colloidal gold and biotin) and storage on antibody microarray sensitivity. The results displayed that the sensitivities of antibody microarray based on agarose gel modified slides with Cy3/HRP conjugated anti-WSSV or anti-LCDV MAbs as detection antibody were 0.31μg/ml for WSSV and 0.55μg/ml for LCDV, which was higher than the ones prepared by other supports or markers. In WSSV concentration range from 0.62 to 9.9μg/ml, LCDV range from 0.55-17.56μg/ml, signal value and logarithmic virus concentration showed a good linear relationship. The correlation coefficients were 0.98925 and 0.9900 respectively. The sensitivity of antibody microarray can be improved to 25 ng/ml or WSSV and 70 ng/ml for LCDV by biotin-streptavidin system to amplify signal value.The signal value of antibody microarray decreased due to the apparent rising of background after 4 months storage at -20°C by using Cy3-conjugated anti-WSSV or anti-LCDV MAbs as detection antibody, which resulted in the sensitivity decrease. When HRP-conjugated anti-WSSV or anti-LCDV MAbs was used as detection antibody, the sensitivity of antibody microarray was still 0.15μg/ml for WSSV while the sensitivity declined to 1.1μg/ml for LCDV after 12-month storage at -20°C.5. Antibody microarrays were applied in samples of diseased shrimp or fish, and results of the antibody microarray with enzyme linked immunosor-bent assay (ELISA) for WSSV/LCDV detection were in 100% concordance and the correlation coefficient (r) was 0.9853/0.9803. These results demonstrate that the antibody microarray can detect the pathogen accurately.6. For the requirements of on-spot detection of aquatic animals’virus, we developed the antibody microarray combining the advantages of the specificity of ELISA, sensitivity and high-throughput of microarray with HRP-conjugated anti-WSSV or anti-LCDV MAbs as detection antibody. The detection results can be read by naked eyes and the detection can be performed on-field conveniently. Detection results of antibody microarray showed 100% concordance with ELISA and >98% concordance with indirect immunofluorescence assay technique (IIFA). In a certain concentration range, signal value and logarithmic virus concentration showed a good linear relationship with which relative quantitative detection of the virus could be performed. The correlation coefficients were 0.9505 and 0.9567 respectively. These merits make it practical in diagnostic and epidemiological studies on WSSV in shrimp or LCDV in fish aquaculture and potentially in other sea animals, in a high efficiency manner.The preparation of antibody microarray system can expand to other aquatic animal virus detection. The antibody microarrays developed for WSSV/LCDV detection can detect multiple samples simultaneously and conveniently. The detection operation with antibody microarray is simple, fast, convenient, accurate, and easy up-scaling to high-throughout at low sample consumption and thus low cost. No expensive equipment was necessary. The antibody microarray provides an effective platform for aquatic animals’pathogen detection and has extensive prospect in disease surveillance and epidemiological studies in aquaculture especially during quarantine inspection on import/export of aquatic goods.更多还原