对虾白斑症病毒和鱼类淋巴囊肿病毒检测抗体芯片的制备与应用
|
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。
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.
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。
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.
引文
1. Alonso M, Rodriguez S, Perez-Prieto SI, 1997. A continuous cell line from the cultured marine fish gilt-head seabream (Sparua aurata L). Aquaculture, 150: 143-153.
2. Alexandre I, Hamels S, Dufour S, et al., 2001. Colorimetric silver detection of DNA microarrays. Anal. Biochem., 295 (3): 1- 8.
3. Al-Harbi AH, Truax R, Thune RL, 2000. Production and characterization of monoclonal antibodies against tilapia Oreochromis niloticus immunoglobulin. Aquaculture, 188: 219-227.
4. Angenendt P, Glokler J, Murphy D, et al., 2002. Toward optimized antibody microarrays: a comparison of current microarray support materials. Anal. Biochem., 309: 253-260.
5. Arenkov P, Kukhtin A, Gemmell A, et al., 2000. Protein microchips: Use for immunoassay and enzymatic reactions. Anal. Biochem., 278(2): 123-131.
6. Arkush KD, McNeiill C, Hedrick RP, 1992. Production and initial characterization of monoclonal antibodies against channel catfish virus, the causative agent of channel catfish virus disease. J. Aaquat. Anim. Health., 4(2): 81-89.
7. Armstrong RD, Ferguson HW, 1989. Systemic viral disease of the chromide cichlid Etroplus marculatus. Dis. Aquat. Org., 7: 155-157.
8. Benjamin T, Houseman, Mrksich M, 2002. Towards quantitative assays with Lveptide chips: a surface engineering approach. Trends Biotechnol, 20(7): 98-90.
9. Bj(?)rck L, Kronvall G, 1984. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. 133(2): 969-974.
10. Block B, Larsen JL, 1993. An iridovirus-like agent associated with systemic infection in cultured turbot Scophalumus maximus fry in denmark. Dis. Aquatic. Org., 15: 235-240.
11. Bonami JR, Lightner DV, et al., 1992. Partial characterization of a togavirus (LOVV) associated with histopathological changes of the lymphoid organ of penaeid shrimp. Dis. Aquat. Org., 14(2): 145-152.
12. Borrebaec CAK, 2000. Immunol. Today, 21: 379-382.
13. Brun MP, Bischof L, Garbay C, 2004. A very short route to enantiomerically pure coumarin-bearing fluorescent amino acids. Angew. Chem., Int. Ed., 43(26): 3432-3436.
14. Caipang CM, Haraguchi I, Ohira T, Hirono I, Aoki T, 2004. Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP). J. Virol. Methods, 121: 155-161.
15. Cano I, Alonso MC, Garcia-Rosado E, Saint-Jean SR, Castro D, Borrego JJ, 2006. Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt-head seabream (Sparus aurata, L.) using an immunoblot technique. Vet. Microbiol., 113: 137-141.
16. Cano I, Ferro P, Alonso MC, et al., 2007. Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species. J. Appl. Microbiol., 102:32-40.
17. Chang SF, Ngoh GH, Kueh LFS, et al., 2001. Development of a tropical marine fish cell line from Asian seabass (Lates calcarifer) for virus isolation. Aquaculture, 192: 133-145.
18. Cheng SF, Zhan WB, Xing J, Sheng XZ, 2006. Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China. J. Virol. Methods, 135: 173-180.
19. Chua FHC, Ng ML, Loo J, et al., 1994. Investigation of outbreaks of a novel disease,“Sleepy Grouper disease’, affecting the brown-spotted grouper, Epinephelus tauvina Forskal. J. Fish. Dis., 17: 417-427.
20. Corber V, Zuprizal, Shi Z, 2001. Experimental infection of European crustaceans with white spot syndrome virus (WSSV). J. Fish Dis., 24: 377-382.
21. Cortez SMM, Villanueva RA, Jashes M, et al., 2009. Molecular characterization of IPNV RNA replication intermediates during the viral infective cycle. Virus Research, 144(1-2): 344-349.
22. Cowley JA, Dimmock CM, 1999. Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses. Dis. Aquat. Org., 36(2): 153-157.
23. Darai G, Delius H, Clarke J, et al., 1985. Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus. Virology. 146: 292-301.
24. Davis PJ, et al., 1994. The detection of infectious pancreatic necrosis virus in asymptomatic carrier fish by an integrated cell-culture and ELISA technique. J. Fish Dis., 17: 99-110.
25. De Ceuninck F, Dassencourt L, Anraet P., 2004. The inflammatory side of human chondrocytes unveiled by antibody microarrays. Biochem. Biophys. Res. Commun., 323(3): 960-969.
26. Deering RE, et al., 1991. Development of a biotinylated DNA probe for detection and identification of infectious hematopoietic necrosis virus. Dis. Aqua. Organ., 11(1): 57-65.
27. Dixon P, Vethaak D, Bucke D, Nicholson M, 1996. Preliminary study of the detection of antibodies to lymphocystis disease virus in flounder, Platichthys flesus L., exposed to contaminated harbour sludge. Fish Shellfish immunol., 6(2): 123-133.
28. Dolores JC, 2000. Protein arrays: a high-throughput solution for proteomics research? Trends in Biotechnology, 18: 47-51.
29. Dopazo CP, et al., 1994. Use of cloned cDNA probes for diagnosis of infectious pancreatic necrosis virus infections. J. Fish Dis. 17: 1-16.
30. Drolet S, Chiou PP, Heidel J, et al., 1995. Detection of truncated virus particles in a persistent RNA virus infect ion in vivo. Virol., 69: 2140-2147.
31. Du H, Yang W, Xing W, et a1., 2005. Parallel detection and quantification using nineimmunoassays in a protein microarray for dmg from serum samples. Biomed Microdevices, 7(2): 143-146.
32. Du WD, Xu ZS, Ma XL, et al., 2003. Biochip as a potential platform of serological interferonα2b antibody assay. J. Biotechnol., 106: 87-100.
33. Durand S, Lightner DV, Nunan LM, et al., 1996. Application of geneprobes as a diagnostic tools for white spot baculovirus (WSBV) of penaeid shrimp. Dis. Aquat. Org., 27: 59-66.
34. Eric WO, James M, Michael PD, et al., 2005. Comparison of antibody array substrates and the use of glycerol to normalize spot morphology. Exp. Mol. Pathol., 79: 206-209.
35. Essbauer S, Fischer U, Bergmann S, Ahne W, 2004. Investigations on the ORF 167L of Lymphocystis Disease Virus (Iridoviridae). Virus Gene, 28(1): 19-39.
36. Fujian N, 1988. Vccination against apring viraemia of carp. In“fish vaccination”(ed. By A. E. Ellis). Academic Press, London. PP. 204-215.
37. Funovics M, Weissleder R, Tung CH, 2003. Protease sensors for bioimaging. Anal. Bioanal. Chem., 377(6): 956-963.
38. Garcia-Rosado E, Castro D, Cano I, et al., 2002. Serological techniquea for detection of lymphocystis in fish. Aquat. Living Reour., 15(3): 179-185.
39. Gehring AG, Albin DM, Bhunia AK, et al., 2006. Antibody microarray detection of Escherichia coli O157:H7 quantification, assay limitations, and capture efficiency. Anal Chem., 78: 6601-6607.
40. González I, et al., 1999. Rapid enumeration of Escherichia coli in oysters by a quantitative PCR-ELISA. J. Appl. Mcrobiol., 86(2): 231-236.
41. González SF, Krug MJ, Nielsen ME, et al., 2004. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray. J. Clin. Microbiol. 42 (4): 1414-1419.
42. Gou DF et al., 1991. Detection of salmonid herpes virus (Oncorhynchus masou virus) in fish by southern-blot technique. J.Vet. Med. Sci., 531: 43-48.
43. Grant SK, Sklar JG, Cummings RT, 2002. Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates. J. Biomolecular Screening, 7(6): 531-540
44. Gregory A, Munro LA, Wallace IS, et al., 2007. Detection of infectious pancreatic necrosis virus (IPNV) from the environment in the vicinity of IPNV-infected Atlantic salmon farms in Scotland. J. Fish Dis., 30(10): 621-630.
45. Gunimaladevi I, Kono T, Venugopal MN, Sakai M, 2004. Detection of koi herpesvirus in common carp. Cyprinus carpio L., by loop-mediated isothermal amplification. J. Fish. Dis., 27: 583–589.
46. Gypi SP, Rochon Y, Franza BR, et al., 1999. Correlation between protein and mRNAabundance in yeast. Mol. Cell Biol., 19(3): 1720-1730.
47. Haab BB, Dunham MJ, Brown PO, 2001. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biology, 2(2):1-13
48. Haab BB, 2001. Advances in protein microarray technology for protein expression and interaction profiling. Curr. Opin. Drug Discov, Devet. 4(1): 116-123.
49. Hasson KW, Lightner DV, Poulos QT., et al., 1995. Taura syndrome in Penaeus vannamen. demonstration of a viral etiology. Dis Aquat Ory., 13: 115-126.
50. He JG, Zen GK, Weng SP, et al., 2002. Experimental transmission, pathogenicity and physical chemical properties of infectious spleen and kidney necrosis virus (ISKNV). Aquaculture, 204: 11-24.
51. Hu GB, Cong RS, Fan TJ, Mei XG, 2004. Induction of apoptosis in floundergill cell line by lymphocystis disease virus infection. J. Fish Dis., 27: 657-662.
52. Holgate CS, 1983. Immunogold-silver staining: new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem., 31(7): 938-944.
53. Huang RP, Huang R, Fan Y, 2001. Simultaneous detection of multiple cytokines from conditioned media and patient’s sera by an antibody-based protein array system. Anal. Biochem., 29(4): 55-62.
54. Huang RP. 2001. Detection of multiple proteins in an antibody-based protein microarray system. J. Immunol. methods, 255(1-2): 1-13.
55. Huang R, Lin Y, Shi Q, et a1., 2004. Enhanced protein profiling arrays with ELISA-based amplification for high-throughput molecular changes of tumor patients’plasma. Clin Cancer Res, 10(2): 598-609.
56. Huang XX, Lu CP, 2006. Genome Sequencing and Structure Prediction of ZHZC3 Strain of Taura Syndrome Virus Isolated in China. Virologica Sinica., 21(3): 267-272.
57. Iwamoto R, Hasegama O, Lapatra S, et al., 2002. Isolation and characterization of the Japanese flounder (Paralichthys olivaceus) lymphocystis disease virus. J. Aquat. Animal Health, 14: 114 -123.
58. Jeong JB, Park KH, Kim HY, et al., 2004. Multiplex PCR for the diagnosis of red sea bream iridovirus isolated in Korea. Aquaculture, 235: 139-152.
59. Jeremy CM, Zhou HP, Joshua K, et al., 2003. Antibody microarray profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers. Proteomics. 3: 56-63.
60. Jiang L, Yu ZB, Du WD, et al., 2008. Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections. Biosens. Bioelectron. 24: 376-382.
61. Jory DE, Dixon HM, 1999. Shrimp white spot virus in the Western Hemisphere. Aquacure. 25: 83-89.
62. Kakizaki E, et al., 1996. Detection of bacterial antigens u-sing immuno-PCR. Appl. Mcrobio., 23 (2): 101-103.
63. Kasai H, Yosimizu M, 2001. Establishment of two Japanese flounder Embryo cell lines. Bull. Fish. Sci., Hokkaido Univ., 52(2): 67-70.
64. Katarzyna D, Levi AG, Claudia P, 2007. A comparative analysis of polyurethane hydrogel for immobilization of IgG on chips. Anal. Chim. Acta., 592: 132-138.
65. Kimura T, Yoshimizu M, Gorie S, 1986. A new rhabdovirus isolated in Japan from cultured hirame (Japanese flounder) Paralichthys olivaceus and ayu Plecoglossus altivelis. Dis. Aquat. Organ., 1: 209-217.
66. Kitamura SI, Jung SJ, Oh MJ, 2006. Differentiation of lymphocystis disease virus genotype by multiplex PCR. J. Microbiol., 44: 248-253.
67. Kono T, Savan R, Sakai M. et al., 2004. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. Virol. Methods, 115(1): 59-65.
68. Kumagai A, Takahashi K, Fukuda H, 1997. Infection source of herpesvirus disease in coho salmon culture and its control. Fish Pathol., 32: 103-108.
69. Kusnezow W, Hoheisel JD, 2003. Solid supports for microarray immunoassays. J. Mol. Recognit., 16(4): 165-176.
70. Lai YS, Chiu HC, 2001. In vnitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus(YGNNV) and immunolocalization of vitus infection in yellow grouper, Epinephelus awoara. J. Fish Dis. 24, 237-244.
71. LaPatra SE, Roberti KA, Rohovec JS, Fryer JL, 1989. Fluorescent antibody test for the rapid diagnosis of infectious hematopoietic necrosis. J. Aquat. Anim. Health, 1: 29-36.
72. Lee Y, Lee EK, Cho YW, et al., 2003. Proteochip: a highly sensitive protein microarray, prepared by a novel method of protein immobilization for application of protein-protein interactions studies. Proteomics. 3(12): 2289-2304.
73. Levit-Binnun N, LindnerAB, Zik O, et al., 2003. Quantitative detection of protein arrays. Anal. Chem., 75(6) : 1436-1441.
74. Li Q, Yue ZQ, Liu H, et al., 2010. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus. J. Virol. Methods, 163(2): 378-384.
75. Liang RQ, Tan CY, Ruan KC., 2004. Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement. J. Immun. Methods, 285 (3): 157 - 163.
76. Lightner DV, 1996. A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. Section 3: Viruses, World Aquaculture Society. Baton Rouge,Louisiana, USA.
77. Lightner DV, Redman RM, 1981. A baculovirus-caused disease of the penaeid shrimp, Penaeus monodon. J. Invertebr. Pathol., 38: 299-302.
78. Lightner DV, Redman RM, Bell TA, 1983. Infectious hypodermal and hematopoietic necrosis, a newly recognized virus disease of penaeid shrimp. J. Invertebr. Pathol., 42: 62-70.
79. Lightner DV, Redman KM., 1998. Shrimp disease and current diagnostic methods. Aquaculture, 164 (1-4): 201-220.
80. Lightner DV, Poulos BT, Bruce L, et al. New developments in penaeid viology: Application of Biotechnology in research disease diagnosis for shrimp viruses of concern in the Americas. In: Fulks, W. & Main, K. L., (eds.), Disease of cultured Penaeid shrimp in Asia and the United states. Honolulu, Hawaii, 1992, 233-253.
81. Liu Z, Teng Y, Xie X, et al., 2008. Development and evaluation of a one-step loop-mediated isothermal amplification for detection of spring viraemia of carp virus. J. Appl. Microbiol., 105: 1220-1226.
82. Lo CF, Ho CH, Peng SE, et al., 1996. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis. Aquat. Organ., 27: 215-222.
83. Lvov Y, Mohwald H, 2000. Protein architecture: interfacing molecular assemblies and immobilization biotechnology. Marcel Dekker, New York. p. 193-227.
84. MacBeath G, Schreiber SL., 2000. Printing proteins as microarrays for high-throughput function determination. Science, 289(5485): 1760-1763.
85. Makesh M, Koteeswaran A, Daniel JCN, et al., 2006. Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb. Aquaculture, 261: 64-71.
86. Mari J, Poulos BT, Lightner DV, Bonami JR, 2002. Shrimp Taura syndrome virus: genomic characterization and similarity with members of the genus Cricket paralysis-like viruses. J. Gen. Virol, 84( 4) : 915-926.
87. Masanori YY, 1991. Immobilization of ultra-thin layer of monoclonal antibody on glass surface. J. Chromatogr. B, 566: 361-368.
88. McKinney MM, Parkinson A, 1987. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J. immuno. methods, 96(2): 271-278.
89. Medina DJ, et al., 1992. Dis. Aquat. Organ., 13(2): 147-150.
90. Miller JC, Zhou H, et a1., 2003. Antibody microarray profiling of human prostate cancer sera: antibody sreening and identification of postate biomarkers. Proteomics, 3(1): 56-63.
91. Misao, Arimoto, et al., 1992. Detection of Striped Jack Nervous Virus (SJNNV) by Enzyme-Linked Immunosobent Assay (ELISA). Fish Pathol., 27(4):191-195.
92. Miyazaki T, Egusa S, 1972. On the lymphocystis disease in cultured sea bass (Lateolabraxjaponicus, Covier and Valenci). Fish Pathol., 6: 83-89.
93. Mourton C, et al., 1992. J. Clin. microbio., 30(9): 2338-2345.
94. Mrikin CA, Lestinger RL, Mucic RC, et al., 1996. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature, 382(6592): 607-609.
95. Nigrelli RF, Ruggieri GD. Studies in virial disease of fishes. Spontaneous and experimentally induced cellular hypertrophy lymphocystis disease in fishes of the New York Aquarium with a report of new cases and an annotated bibliography (1874-1965). Zoologica, 1965, 50: 83-96.
96. Notomi T, Okayama H, Masubuchi H, et al., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63.
97. Nunan LM, Poulos BT, Lightner DV., 1998. The detection of White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) in imported commodity shrimp. Aquaculture. 160: 19-30.
98. Oh MJ, Choi TJ, 1998. A new rhabdovirus (HRV-like) isolated in Korea from cultured Japanese flounder Paralichthys olivaceus. J. Fish Pathol. 11: 129-136.
99. Ozgur G, Ediz C, Ugur S, et al., 2007. Sandwich-type, antibody microarrays for the detection and quantification of cardiovascular risk markers. Sens. Actuators. B 125: 581-588.
100. Panagiota SP, Margarita C, Antonios MD, et al., 2007. A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on dilicon chips. Biosens. Bioelectron., 22: 1994-2002.
101. Pavel A, Alexander K, Anne G, et a1., 2000. Protein Microchip: Use for Immunoassay and Enzymatic Reactions. Anal. Biochem., 278: 23-31.
102. Peluso P, Wilson DS, Do D, et a1., 2003. Optimizing antibody immobilization strategies for the construction of protein microarrays. Anal. Biochem., 312: 113-124.
103. Poulos BT. Use of t he PCR. Detect viral infections in penaeid shrimp. Browdy C L (Eds), Swimming through troubled water proceeding of the special session on shrimp farming, aquaculture’. World Aquaculture society, Baton Rouge, LA, 1995. 238-239.
104. Pozet F, Morand M, Torhy A, et al., 1992. Isolation and preliminary characterization of a pathogenic icosahedral deoxyribovirus from the catfish Ictalurus melas. Dis. Aquat. Org., 14: 35-42.
105. Ristow SS, Arnzen JM, 1989. Development of monoclonal antibodies that recognize a type- specific and a common epitope on the nucleoprotein of infectious hematopoietic necrosis virus. J. Aquat. Anim. Health, 1: 119-125.
106. Robles-Sikisaka R, Hasson KW, Garcia DK, et al., 2002. Genetic variation and immunohistochemical differences among geographic isolates of Taura syndrome virus of penaeid shrimp. J. Gen. Virol., 83(12): 3123-3130.
107. Robinson WH, DiGcanaro C, Hueber W, et a1., 2002. Autoantigen microarrays for multiplex characteri-zation of autoantibody responses. Nat. Med., 8(3): 295-301.
108. Rodger, et al., 1997. Systemic iridovirus infection in freshwater angelfish, Pterophllum scalare (Lichtenstein). J. Fish. Dis., 20: 69-72.
109. Ronan Q, The′re`se C, Jacques M, et al., 2002. White spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus simultaneous diagnosis by miniarray system with colorimetry detection. J. Virol. Methods, 105: 189-196.
110. Rubina AY, Dementieva EI, Stomakhin AA, et al., 2003. Hydrogel-based protein microchips: manufacturing, properties, and applications. Biotechniques, 34(5): 1008-1014.
111. Sano T, Fukuda H, 1987. Principal microbial disease of mariculture in Japan. Aquaculture, 67: 59-70.
112. Sasaki E, Kijima H, Nishimatsu H, et al., J. Am. Chem. Soc., 2005, 127(11): 3684-3685.
113. Schaeferling M, Kambhampati D. 2004. Protein Microarray Surface Chemistry and Coupling Schemes. In: Protein Microarray Technology. Edited by Dev Kambhampati. 11-38.
114. Shannon LSS, Amanda MW, Cheryl LB, et al., 2007. Evaluation of surface chemistries for antibody microarrays. Anal. Biochem., 371: 105-115.
115. Shi CY, Wang YG, Yang SL, et al., 2004. The first report of an iridovirus like gent infection in farmed turbot, Scophthalmus maximus, in China. Aquaculture, 236: 11-25.
116. Soliman H, El-Matbouli M, 2006. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS). Vet. Microbiol., 114: 205-213.
117. Sorimachi M, Hara T, 1985. Characteristics and pathogenicity of a virus isolated from yellowtail fingerlings showing ascites. Fish Pathol., 19: 231-238 (in Japanese with English abstract).
118. Stewart TGB, Fiona K, Nichola OL, et al., 2008. A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood. Anal. Biochem., 382: 9-15.
119. Sun Y, Jiang Y, Liu H,et al., 2009. The isolation and characterization of a rhabdovirus from stone flounder, Kareius bicoloratus. Chin. J. Vet. Sci., 29: 277-282.
120. Sun YJ, Yue ZQ, Liu H, et al., 2010. Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR. J.Virol. Methods, 169: 391-396
121. Sun ZF, Hu CQ, Ren Ch, et al., 2006. Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification. Virol. Methods, 131(1): 41-46.
122. Tang ZM, Mei Q, Zhang CX, Zhu Y, Lu ZH, 2002. A comparative study of protein arrays immobilized on different substrates. In: Chinese Materials Research Society ed. Advanced Nanomaterials and Nanodevices. Presented at the 8th International Conference on Electronic Materials. Xi’an, China. p.359-369.
123. Tidona CA, Darai G, 1999. Lymphocystis disease virus (Iridoviridae), p. 908–911. In A. Granoff and R. G. Webster (ed.), Encyclopedia of virology. Academic Press, New York.
124. Tung CH, Gerszten RE, Jaffer FA, et al., 2002. A novel near-infrared fluorescence sensor for detection of thrombin activation in blood. Chembiochem, 3(2-3): 207-211.
125. Vamum SM, Woodbury RL, Zangar RC, 2004. A protein microarray ELISA for Screening biological fluids. Methods Mot. Biol., 278: 161-172
126. Victor NM, Tamara YM, 2006. Active bead-linked immunoassay on protein microarrays. Anal. Chim. Acta., 564: 40-52.
127. Walker R. 1962. Fine structure of lymphocystis disease virus in fish. Virology, 18:503-505.
128. Wang XJ, Zhan WB, 2006a. Development of an immunochromatographic test to detect White Spot Syndrome Virus of shrimp. Aquaculture, 255: 196-200.
129. Wang XJ, Zhan WB, Xing J., 2006b. Development of dot-immunogold filtration assay to detect white spot syndrome virus of shrimp. J. Virol. Methods, 132: 212-215.
130. Wang YC, Lo CF, Chang PS, Kou GH, 1998. White spot syndrome associated virus (WSSV) infection in cultured and wild decapods in Taiwan. Aquaculture, 164: 221-231.
131. Wang Z, Lee J, Cossins AR, et al., 2005. Microarray-based detection of protein binding and functionality by gold nanoparticle probes. Anal. Chem., 77(17): 5770-5774.
132. Wolf K, Gravell M, Malsberger RG, 1966. Lymphocystis virus: Isolation and propagation in centrarchid fish cell lines. Science, 151: 1004-1005.
133. Woodbury RL, Varnum SM, Zangar RC, et al., 2002. Elevated HGF levels in sera from breast cancer patients detected using a protein microarray ELISA. J. Proteome Research, 1(3): 233-237.
134. Xing J, Zhan WB, Zeng XH, Cheng SF, 2006. Detection of lymphocystis disease virus infection to flounder gill cells in vitro by monoclonal antibodies. High Technology Letters, 12(1): 103-108.
135. Xu RZ, Gan XX, Fang YM, et al., 2007. A simple, rapid, and sensitive integrated protein microarray for simultaneous detection of multiple antigens and antibodies of five human hepatitis viruses (HBV, HCV, HDV, HEV, and HGV). Anal. Biochem., 362: 69-75.
136. Yegneswaran S, Fernandez JA, Grifin JH, et al., 2002. Factor Va increases the affinity of factor Xa for prothrombin: a binding study using a novel photoactivable thiol-specific fluorescent probe. Chem. Biol., 9(4): 485-494.
137. Yu W, Gu N, Zhang H Q, et al. 2004. Microarray preparation based on oxidation ofagarose-gel. Sensors and Actuators B, 98: 83-91.
138. Zammatteo N, Jeanmart L, Hamels S, et al., 2000. Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays. Anal. Biochem., 280(1): 143-50
139. Zhan WB, Wang YH, John LF, et al., 1998. White Spot Syndrome Virus Infection of Cultured Shrimp in China. J. Aqua. Animal Health., 10: 405-410.
140. Zhan W B, et al., 1999. Using developed monoclonal antibodies for detection of Penaeus chinensis WSSV infection. In: Proceeding of International Symposium on Progress and Prospect of Marine Biotechnology. Qingdao. 156-161.
141. Zhan WB, Li YQ, Sheng XZ, et al., 2010. Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China. Chinese Journal of Oceannology and Limnology. 28(6): 1214-1220.
142. Zhang QY, Ruan HM, Li ZQ, et al., 2003. Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines. Dis. Aquat. Org., 57: 27-34.
143. Zhou J, Larissa B, Pauline YH, et al., 2010. Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing. J. Immunol. Methods, 355: 40-51.
144. Zhu H, Snyder M, 2003. Protein chip tecimology. Curr. Opin. Chem. Biol., 7(1): 55-63.
145.陈信忠,龚艳清, 2006.鱼类病毒性神经坏死病研究进展.生物技术通报. S1: 141-146.
146.黄倢,宋晓玲,于佳等, 1995.杆状病毒的皮下及造血组织坏死-对虾爆发性流行病的病原和病理学.海洋水产研究, 161: 110-115.
147.黄倢,杨丛海,于佳等, 1995b. T-E染色法用于对虾暴发性流行病的现场快速诊断.海洋科学1: 29-34.
148.黄艳青,王桂堂,孙军等, 2003.黄颡鱼血清免疫球蛋白的纯化及分子量的初步测定.水生生物学报, 27(6): 654-656.
149.黄晓峰,张远强,张英起, 2004.荧光探针技术.北京:人民军医出版社, 404-408.
150.江凌晓,郭兆彪,陈泽良等, 2004.蛋白质芯片制作条件的优化.第一军医大学学报,24(11):1230-1232
151.姜红,李月红,吴东明,付云红, 2010.鱼类传染性造血器官坏死病临床诊断及检测.中国水产, 11:53-57.
152.江育林,李正秋, 1991.病毒感染的草鱼细胞产生类干扰素物质的研究.病毒学报, 7:30-35.
153.江育林, 1990.用酶联免疫吸附试验快速检测虹蹲的传染性胰脏坏死病病毒.水生生物学报, 14(3): 276-279.
154.江育林等, 1993,两种快速检测技术实验条件的探索及特性比较,第三次鱼病研讨会论文摘要汇编.
155.雷质文,史成银,莫照兰, 2002.人工感染白斑综合征病毒的克氏原螯虾的病理学试验.中国兽医科技, 32: 23-25.
156.李永芹.养殖鱼类病原菌检测芯片技术的建立. [博士学位论文].青岛:中国海洋大学水产学院, 2010.
157.李贵生,何建国,吴冰等, 2001.斑节对虾杆状病毒在感染对虾中肠腺中的分布.水产学报,25(2):141-147.
158.吕宏旭,汪岷,李红岩等, 2003.利用牙鲆鳃细胞系分离和培养淋巴囊肿病毒.青岛海洋大学学报, 33(2): 233-239.
159.刘志红,何为,韩旭等, 2007.琼脂糖凝胶蛋白芯片片基的制备及应用.分析化学, 35(5): 775-778.
160.刘荭,范万红,史秀杰等, 2006.国内养殖鱼类和进境鱼卵中传染性造血器官坏死病毒(IHNV)的检测及基因分析.华中农业大学学报. 25(5): 544-549.
161.黎小正,韦信贤,吴祥庆等, 2008.对虾Taura综合征病毒RT-PCR检测方法的改进及应用.中国预防兽医学报. 17(5): 525-529.
162.林天龙,陈强,俞伏松等, 2001.欧洲鳗血清免疫球蛋白纯化及部分特性分析.水产学报, 25(1): 52-57.
163.林炳承,秦建华, 2005.微流控芯片实验室.色谱. 23 (5): 456- 463.
164.李惠芳,刘荭,吕建强等, 2008. TaqMan实时荧光PCR快速检测斑点叉尾鮰病毒.长江大学学报,自然科学版. 5(1): 42-46.
165.马立人,蒋中华主编. 2002.生物芯片.第2版,北京:化学工业出版社. 40-56.
166.苗宏志,童裳亮,徐斌等, 2000.利用对虾原代细胞增殖对虾杆状病毒HHNBV的研究.生物工程学报. 16(2): 221-223.
167.曲径,沈海平,李笑刚等, 2001.威海地区养殖牙鲆鱼淋巴囊肿病流行病学调查.检验检疫科学, 11(6): 34-35.
168.孙广瑞,李小兵,王哲, 2007.制备分泌单克隆抗体杂交瘤细胞小鼠腹水的方法.中国现代医生. 45(5): 3-4.
169.绳秀珍.鱼类淋巴囊肿病毒的流行病学研究. [博士学位论文].青岛:中国海洋大学水产学院, 2006.
170.石锐,孙凯,王秀荣等, 2005.生物芯片在疾病诊断中的应用.畜牧兽医科技信息. 5: 7-8.
171.沈明山,罗文新,陈晋安等, 2000.厦门地区杂色花蛤的病毒观察与检测.海洋通报. 19(1): 93-96.
172.史成银,王印庚,黄倢等, 2005.大菱鲆红体病虹彩病毒ATPase基因的克隆与序列分析.高技术通讯. 5(15): 91-95.
173.孙修勤,张进兴, 1998.中国对虾肝胰腺的细小病毒病的直接荧光抗体诊断研究.黄渤海海洋. 16(4): 48-53.
174.王崇明,宋微波, 2002.栉孔扇贝一种球形病毒的分离纯化及其超微结构观察.水产学报. 26(2): 180-184.
175.王江勇,陈毕生,冯娟, 2000.杂色鲍裂壳病球状病毒的初步观察.热带海洋. 19(4): 82-85.
176.王炜,陈延,柯丽华等, 1990.草鱼出血病病毒武汉南湖株的精细结构与基因组及其多肽的研究.中国病毒学, 6(1): 41-49.
177.王艳,何为,刘志红, 2002.蛋白芯片的研究进展及其临床应用.国外医学:微生物学分册. 25(2): 7-9.
178.王艳霞,李宁,傅星等, 2003.原子力显微镜在生物力的测定应用.微电子技术. 7-8: 228~230。
179.许拉,黄倢,杨冰, 2008.病原检测基因芯片应用及在水产病害检测的前景.海洋水产研究. 29(1): 109-114.
180.谢树涛,何建国,杨晓明等, 2001.套式PCR检测斑节对虾白斑症病毒(WSSV).青岛海洋大学学报. 31(2): 220-224.
181.熊炜,邱璐,李健等, 2007. Real-time PCR方法和PCR方法检测虾白斑综合征病毒.中国预防兽医学报. 29 (2): 138-141.
182.颜桦.抗体芯片基本技术平台的建立及其在肝脏蛋白质组学研究中的应用。[硕士学位论文].陕西:西北大学, 2006.
183.曾伟伟,王庆,石存斌等, 2010.免疫学和分子生物学技术在水产动物疾病诊断中的应用.动物医学进展. 31(6): 111-117.
184.战文斌主编,水产动物病害学.青岛:中国农业出版社. 2004.
185.张奇亚,李正秋, 2001.胭脂鱼弹状病毒包涵体在培养细胞中的形成.中国兽医学报. 21(1): 37-40.
186.张永嘉,郭青,吴泽阳, 1997.云纹石板鱼淋巴囊肿病变过程的超微研究.海洋与湖沼. 28: 406-410.
187.朱建中,陆承平, 2001.对虾白斑综合征病毒在螯虾动物模型的感染特性.水产学报. 25: 47-58.
188.左小霞,高志贤,曹巧玲.葡萄球菌肠毒素A、B检测的免疫芯片技术研究.中国卫生检验杂志, 2008, 18(12): 2483-2488.
2. Alexandre I, Hamels S, Dufour S, et al., 2001. Colorimetric silver detection of DNA microarrays. Anal. Biochem., 295 (3): 1- 8.
3. Al-Harbi AH, Truax R, Thune RL, 2000. Production and characterization of monoclonal antibodies against tilapia Oreochromis niloticus immunoglobulin. Aquaculture, 188: 219-227.
4. Angenendt P, Glokler J, Murphy D, et al., 2002. Toward optimized antibody microarrays: a comparison of current microarray support materials. Anal. Biochem., 309: 253-260.
5. Arenkov P, Kukhtin A, Gemmell A, et al., 2000. Protein microchips: Use for immunoassay and enzymatic reactions. Anal. Biochem., 278(2): 123-131.
6. Arkush KD, McNeiill C, Hedrick RP, 1992. Production and initial characterization of monoclonal antibodies against channel catfish virus, the causative agent of channel catfish virus disease. J. Aaquat. Anim. Health., 4(2): 81-89.
7. Armstrong RD, Ferguson HW, 1989. Systemic viral disease of the chromide cichlid Etroplus marculatus. Dis. Aquat. Org., 7: 155-157.
8. Benjamin T, Houseman, Mrksich M, 2002. Towards quantitative assays with Lveptide chips: a surface engineering approach. Trends Biotechnol, 20(7): 98-90.
9. Bj(?)rck L, Kronvall G, 1984. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. 133(2): 969-974.
10. Block B, Larsen JL, 1993. An iridovirus-like agent associated with systemic infection in cultured turbot Scophalumus maximus fry in denmark. Dis. Aquatic. Org., 15: 235-240.
11. Bonami JR, Lightner DV, et al., 1992. Partial characterization of a togavirus (LOVV) associated with histopathological changes of the lymphoid organ of penaeid shrimp. Dis. Aquat. Org., 14(2): 145-152.
12. Borrebaec CAK, 2000. Immunol. Today, 21: 379-382.
13. Brun MP, Bischof L, Garbay C, 2004. A very short route to enantiomerically pure coumarin-bearing fluorescent amino acids. Angew. Chem., Int. Ed., 43(26): 3432-3436.
14. Caipang CM, Haraguchi I, Ohira T, Hirono I, Aoki T, 2004. Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP). J. Virol. Methods, 121: 155-161.
15. Cano I, Alonso MC, Garcia-Rosado E, Saint-Jean SR, Castro D, Borrego JJ, 2006. Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt-head seabream (Sparus aurata, L.) using an immunoblot technique. Vet. Microbiol., 113: 137-141.
16. Cano I, Ferro P, Alonso MC, et al., 2007. Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species. J. Appl. Microbiol., 102:32-40.
17. Chang SF, Ngoh GH, Kueh LFS, et al., 2001. Development of a tropical marine fish cell line from Asian seabass (Lates calcarifer) for virus isolation. Aquaculture, 192: 133-145.
18. Cheng SF, Zhan WB, Xing J, Sheng XZ, 2006. Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China. J. Virol. Methods, 135: 173-180.
19. Chua FHC, Ng ML, Loo J, et al., 1994. Investigation of outbreaks of a novel disease,“Sleepy Grouper disease’, affecting the brown-spotted grouper, Epinephelus tauvina Forskal. J. Fish. Dis., 17: 417-427.
20. Corber V, Zuprizal, Shi Z, 2001. Experimental infection of European crustaceans with white spot syndrome virus (WSSV). J. Fish Dis., 24: 377-382.
21. Cortez SMM, Villanueva RA, Jashes M, et al., 2009. Molecular characterization of IPNV RNA replication intermediates during the viral infective cycle. Virus Research, 144(1-2): 344-349.
22. Cowley JA, Dimmock CM, 1999. Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses. Dis. Aquat. Org., 36(2): 153-157.
23. Darai G, Delius H, Clarke J, et al., 1985. Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus. Virology. 146: 292-301.
24. Davis PJ, et al., 1994. The detection of infectious pancreatic necrosis virus in asymptomatic carrier fish by an integrated cell-culture and ELISA technique. J. Fish Dis., 17: 99-110.
25. De Ceuninck F, Dassencourt L, Anraet P., 2004. The inflammatory side of human chondrocytes unveiled by antibody microarrays. Biochem. Biophys. Res. Commun., 323(3): 960-969.
26. Deering RE, et al., 1991. Development of a biotinylated DNA probe for detection and identification of infectious hematopoietic necrosis virus. Dis. Aqua. Organ., 11(1): 57-65.
27. Dixon P, Vethaak D, Bucke D, Nicholson M, 1996. Preliminary study of the detection of antibodies to lymphocystis disease virus in flounder, Platichthys flesus L., exposed to contaminated harbour sludge. Fish Shellfish immunol., 6(2): 123-133.
28. Dolores JC, 2000. Protein arrays: a high-throughput solution for proteomics research? Trends in Biotechnology, 18: 47-51.
29. Dopazo CP, et al., 1994. Use of cloned cDNA probes for diagnosis of infectious pancreatic necrosis virus infections. J. Fish Dis. 17: 1-16.
30. Drolet S, Chiou PP, Heidel J, et al., 1995. Detection of truncated virus particles in a persistent RNA virus infect ion in vivo. Virol., 69: 2140-2147.
31. Du H, Yang W, Xing W, et a1., 2005. Parallel detection and quantification using nineimmunoassays in a protein microarray for dmg from serum samples. Biomed Microdevices, 7(2): 143-146.
32. Du WD, Xu ZS, Ma XL, et al., 2003. Biochip as a potential platform of serological interferonα2b antibody assay. J. Biotechnol., 106: 87-100.
33. Durand S, Lightner DV, Nunan LM, et al., 1996. Application of geneprobes as a diagnostic tools for white spot baculovirus (WSBV) of penaeid shrimp. Dis. Aquat. Org., 27: 59-66.
34. Eric WO, James M, Michael PD, et al., 2005. Comparison of antibody array substrates and the use of glycerol to normalize spot morphology. Exp. Mol. Pathol., 79: 206-209.
35. Essbauer S, Fischer U, Bergmann S, Ahne W, 2004. Investigations on the ORF 167L of Lymphocystis Disease Virus (Iridoviridae). Virus Gene, 28(1): 19-39.
36. Fujian N, 1988. Vccination against apring viraemia of carp. In“fish vaccination”(ed. By A. E. Ellis). Academic Press, London. PP. 204-215.
37. Funovics M, Weissleder R, Tung CH, 2003. Protease sensors for bioimaging. Anal. Bioanal. Chem., 377(6): 956-963.
38. Garcia-Rosado E, Castro D, Cano I, et al., 2002. Serological techniquea for detection of lymphocystis in fish. Aquat. Living Reour., 15(3): 179-185.
39. Gehring AG, Albin DM, Bhunia AK, et al., 2006. Antibody microarray detection of Escherichia coli O157:H7 quantification, assay limitations, and capture efficiency. Anal Chem., 78: 6601-6607.
40. González I, et al., 1999. Rapid enumeration of Escherichia coli in oysters by a quantitative PCR-ELISA. J. Appl. Mcrobiol., 86(2): 231-236.
41. González SF, Krug MJ, Nielsen ME, et al., 2004. Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray. J. Clin. Microbiol. 42 (4): 1414-1419.
42. Gou DF et al., 1991. Detection of salmonid herpes virus (Oncorhynchus masou virus) in fish by southern-blot technique. J.Vet. Med. Sci., 531: 43-48.
43. Grant SK, Sklar JG, Cummings RT, 2002. Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates. J. Biomolecular Screening, 7(6): 531-540
44. Gregory A, Munro LA, Wallace IS, et al., 2007. Detection of infectious pancreatic necrosis virus (IPNV) from the environment in the vicinity of IPNV-infected Atlantic salmon farms in Scotland. J. Fish Dis., 30(10): 621-630.
45. Gunimaladevi I, Kono T, Venugopal MN, Sakai M, 2004. Detection of koi herpesvirus in common carp. Cyprinus carpio L., by loop-mediated isothermal amplification. J. Fish. Dis., 27: 583–589.
46. Gypi SP, Rochon Y, Franza BR, et al., 1999. Correlation between protein and mRNAabundance in yeast. Mol. Cell Biol., 19(3): 1720-1730.
47. Haab BB, Dunham MJ, Brown PO, 2001. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biology, 2(2):1-13
48. Haab BB, 2001. Advances in protein microarray technology for protein expression and interaction profiling. Curr. Opin. Drug Discov, Devet. 4(1): 116-123.
49. Hasson KW, Lightner DV, Poulos QT., et al., 1995. Taura syndrome in Penaeus vannamen. demonstration of a viral etiology. Dis Aquat Ory., 13: 115-126.
50. He JG, Zen GK, Weng SP, et al., 2002. Experimental transmission, pathogenicity and physical chemical properties of infectious spleen and kidney necrosis virus (ISKNV). Aquaculture, 204: 11-24.
51. Hu GB, Cong RS, Fan TJ, Mei XG, 2004. Induction of apoptosis in floundergill cell line by lymphocystis disease virus infection. J. Fish Dis., 27: 657-662.
52. Holgate CS, 1983. Immunogold-silver staining: new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem., 31(7): 938-944.
53. Huang RP, Huang R, Fan Y, 2001. Simultaneous detection of multiple cytokines from conditioned media and patient’s sera by an antibody-based protein array system. Anal. Biochem., 29(4): 55-62.
54. Huang RP. 2001. Detection of multiple proteins in an antibody-based protein microarray system. J. Immunol. methods, 255(1-2): 1-13.
55. Huang R, Lin Y, Shi Q, et a1., 2004. Enhanced protein profiling arrays with ELISA-based amplification for high-throughput molecular changes of tumor patients’plasma. Clin Cancer Res, 10(2): 598-609.
56. Huang XX, Lu CP, 2006. Genome Sequencing and Structure Prediction of ZHZC3 Strain of Taura Syndrome Virus Isolated in China. Virologica Sinica., 21(3): 267-272.
57. Iwamoto R, Hasegama O, Lapatra S, et al., 2002. Isolation and characterization of the Japanese flounder (Paralichthys olivaceus) lymphocystis disease virus. J. Aquat. Animal Health, 14: 114 -123.
58. Jeong JB, Park KH, Kim HY, et al., 2004. Multiplex PCR for the diagnosis of red sea bream iridovirus isolated in Korea. Aquaculture, 235: 139-152.
59. Jeremy CM, Zhou HP, Joshua K, et al., 2003. Antibody microarray profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers. Proteomics. 3: 56-63.
60. Jiang L, Yu ZB, Du WD, et al., 2008. Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections. Biosens. Bioelectron. 24: 376-382.
61. Jory DE, Dixon HM, 1999. Shrimp white spot virus in the Western Hemisphere. Aquacure. 25: 83-89.
62. Kakizaki E, et al., 1996. Detection of bacterial antigens u-sing immuno-PCR. Appl. Mcrobio., 23 (2): 101-103.
63. Kasai H, Yosimizu M, 2001. Establishment of two Japanese flounder Embryo cell lines. Bull. Fish. Sci., Hokkaido Univ., 52(2): 67-70.
64. Katarzyna D, Levi AG, Claudia P, 2007. A comparative analysis of polyurethane hydrogel for immobilization of IgG on chips. Anal. Chim. Acta., 592: 132-138.
65. Kimura T, Yoshimizu M, Gorie S, 1986. A new rhabdovirus isolated in Japan from cultured hirame (Japanese flounder) Paralichthys olivaceus and ayu Plecoglossus altivelis. Dis. Aquat. Organ., 1: 209-217.
66. Kitamura SI, Jung SJ, Oh MJ, 2006. Differentiation of lymphocystis disease virus genotype by multiplex PCR. J. Microbiol., 44: 248-253.
67. Kono T, Savan R, Sakai M. et al., 2004. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. Virol. Methods, 115(1): 59-65.
68. Kumagai A, Takahashi K, Fukuda H, 1997. Infection source of herpesvirus disease in coho salmon culture and its control. Fish Pathol., 32: 103-108.
69. Kusnezow W, Hoheisel JD, 2003. Solid supports for microarray immunoassays. J. Mol. Recognit., 16(4): 165-176.
70. Lai YS, Chiu HC, 2001. In vnitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus(YGNNV) and immunolocalization of vitus infection in yellow grouper, Epinephelus awoara. J. Fish Dis. 24, 237-244.
71. LaPatra SE, Roberti KA, Rohovec JS, Fryer JL, 1989. Fluorescent antibody test for the rapid diagnosis of infectious hematopoietic necrosis. J. Aquat. Anim. Health, 1: 29-36.
72. Lee Y, Lee EK, Cho YW, et al., 2003. Proteochip: a highly sensitive protein microarray, prepared by a novel method of protein immobilization for application of protein-protein interactions studies. Proteomics. 3(12): 2289-2304.
73. Levit-Binnun N, LindnerAB, Zik O, et al., 2003. Quantitative detection of protein arrays. Anal. Chem., 75(6) : 1436-1441.
74. Li Q, Yue ZQ, Liu H, et al., 2010. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus. J. Virol. Methods, 163(2): 378-384.
75. Liang RQ, Tan CY, Ruan KC., 2004. Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement. J. Immun. Methods, 285 (3): 157 - 163.
76. Lightner DV, 1996. A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp. Section 3: Viruses, World Aquaculture Society. Baton Rouge,Louisiana, USA.
77. Lightner DV, Redman RM, 1981. A baculovirus-caused disease of the penaeid shrimp, Penaeus monodon. J. Invertebr. Pathol., 38: 299-302.
78. Lightner DV, Redman RM, Bell TA, 1983. Infectious hypodermal and hematopoietic necrosis, a newly recognized virus disease of penaeid shrimp. J. Invertebr. Pathol., 42: 62-70.
79. Lightner DV, Redman KM., 1998. Shrimp disease and current diagnostic methods. Aquaculture, 164 (1-4): 201-220.
80. Lightner DV, Poulos BT, Bruce L, et al. New developments in penaeid viology: Application of Biotechnology in research disease diagnosis for shrimp viruses of concern in the Americas. In: Fulks, W. & Main, K. L., (eds.), Disease of cultured Penaeid shrimp in Asia and the United states. Honolulu, Hawaii, 1992, 233-253.
81. Liu Z, Teng Y, Xie X, et al., 2008. Development and evaluation of a one-step loop-mediated isothermal amplification for detection of spring viraemia of carp virus. J. Appl. Microbiol., 105: 1220-1226.
82. Lo CF, Ho CH, Peng SE, et al., 1996. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis. Aquat. Organ., 27: 215-222.
83. Lvov Y, Mohwald H, 2000. Protein architecture: interfacing molecular assemblies and immobilization biotechnology. Marcel Dekker, New York. p. 193-227.
84. MacBeath G, Schreiber SL., 2000. Printing proteins as microarrays for high-throughput function determination. Science, 289(5485): 1760-1763.
85. Makesh M, Koteeswaran A, Daniel JCN, et al., 2006. Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb. Aquaculture, 261: 64-71.
86. Mari J, Poulos BT, Lightner DV, Bonami JR, 2002. Shrimp Taura syndrome virus: genomic characterization and similarity with members of the genus Cricket paralysis-like viruses. J. Gen. Virol, 84( 4) : 915-926.
87. Masanori YY, 1991. Immobilization of ultra-thin layer of monoclonal antibody on glass surface. J. Chromatogr. B, 566: 361-368.
88. McKinney MM, Parkinson A, 1987. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J. immuno. methods, 96(2): 271-278.
89. Medina DJ, et al., 1992. Dis. Aquat. Organ., 13(2): 147-150.
90. Miller JC, Zhou H, et a1., 2003. Antibody microarray profiling of human prostate cancer sera: antibody sreening and identification of postate biomarkers. Proteomics, 3(1): 56-63.
91. Misao, Arimoto, et al., 1992. Detection of Striped Jack Nervous Virus (SJNNV) by Enzyme-Linked Immunosobent Assay (ELISA). Fish Pathol., 27(4):191-195.
92. Miyazaki T, Egusa S, 1972. On the lymphocystis disease in cultured sea bass (Lateolabraxjaponicus, Covier and Valenci). Fish Pathol., 6: 83-89.
93. Mourton C, et al., 1992. J. Clin. microbio., 30(9): 2338-2345.
94. Mrikin CA, Lestinger RL, Mucic RC, et al., 1996. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature, 382(6592): 607-609.
95. Nigrelli RF, Ruggieri GD. Studies in virial disease of fishes. Spontaneous and experimentally induced cellular hypertrophy lymphocystis disease in fishes of the New York Aquarium with a report of new cases and an annotated bibliography (1874-1965). Zoologica, 1965, 50: 83-96.
96. Notomi T, Okayama H, Masubuchi H, et al., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, E63.
97. Nunan LM, Poulos BT, Lightner DV., 1998. The detection of White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) in imported commodity shrimp. Aquaculture. 160: 19-30.
98. Oh MJ, Choi TJ, 1998. A new rhabdovirus (HRV-like) isolated in Korea from cultured Japanese flounder Paralichthys olivaceus. J. Fish Pathol. 11: 129-136.
99. Ozgur G, Ediz C, Ugur S, et al., 2007. Sandwich-type, antibody microarrays for the detection and quantification of cardiovascular risk markers. Sens. Actuators. B 125: 581-588.
100. Panagiota SP, Margarita C, Antonios MD, et al., 2007. A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on dilicon chips. Biosens. Bioelectron., 22: 1994-2002.
101. Pavel A, Alexander K, Anne G, et a1., 2000. Protein Microchip: Use for Immunoassay and Enzymatic Reactions. Anal. Biochem., 278: 23-31.
102. Peluso P, Wilson DS, Do D, et a1., 2003. Optimizing antibody immobilization strategies for the construction of protein microarrays. Anal. Biochem., 312: 113-124.
103. Poulos BT. Use of t he PCR. Detect viral infections in penaeid shrimp. Browdy C L (Eds), Swimming through troubled water proceeding of the special session on shrimp farming, aquaculture’. World Aquaculture society, Baton Rouge, LA, 1995. 238-239.
104. Pozet F, Morand M, Torhy A, et al., 1992. Isolation and preliminary characterization of a pathogenic icosahedral deoxyribovirus from the catfish Ictalurus melas. Dis. Aquat. Org., 14: 35-42.
105. Ristow SS, Arnzen JM, 1989. Development of monoclonal antibodies that recognize a type- specific and a common epitope on the nucleoprotein of infectious hematopoietic necrosis virus. J. Aquat. Anim. Health, 1: 119-125.
106. Robles-Sikisaka R, Hasson KW, Garcia DK, et al., 2002. Genetic variation and immunohistochemical differences among geographic isolates of Taura syndrome virus of penaeid shrimp. J. Gen. Virol., 83(12): 3123-3130.
107. Robinson WH, DiGcanaro C, Hueber W, et a1., 2002. Autoantigen microarrays for multiplex characteri-zation of autoantibody responses. Nat. Med., 8(3): 295-301.
108. Rodger, et al., 1997. Systemic iridovirus infection in freshwater angelfish, Pterophllum scalare (Lichtenstein). J. Fish. Dis., 20: 69-72.
109. Ronan Q, The′re`se C, Jacques M, et al., 2002. White spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus simultaneous diagnosis by miniarray system with colorimetry detection. J. Virol. Methods, 105: 189-196.
110. Rubina AY, Dementieva EI, Stomakhin AA, et al., 2003. Hydrogel-based protein microchips: manufacturing, properties, and applications. Biotechniques, 34(5): 1008-1014.
111. Sano T, Fukuda H, 1987. Principal microbial disease of mariculture in Japan. Aquaculture, 67: 59-70.
112. Sasaki E, Kijima H, Nishimatsu H, et al., J. Am. Chem. Soc., 2005, 127(11): 3684-3685.
113. Schaeferling M, Kambhampati D. 2004. Protein Microarray Surface Chemistry and Coupling Schemes. In: Protein Microarray Technology. Edited by Dev Kambhampati. 11-38.
114. Shannon LSS, Amanda MW, Cheryl LB, et al., 2007. Evaluation of surface chemistries for antibody microarrays. Anal. Biochem., 371: 105-115.
115. Shi CY, Wang YG, Yang SL, et al., 2004. The first report of an iridovirus like gent infection in farmed turbot, Scophthalmus maximus, in China. Aquaculture, 236: 11-25.
116. Soliman H, El-Matbouli M, 2006. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS). Vet. Microbiol., 114: 205-213.
117. Sorimachi M, Hara T, 1985. Characteristics and pathogenicity of a virus isolated from yellowtail fingerlings showing ascites. Fish Pathol., 19: 231-238 (in Japanese with English abstract).
118. Stewart TGB, Fiona K, Nichola OL, et al., 2008. A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood. Anal. Biochem., 382: 9-15.
119. Sun Y, Jiang Y, Liu H,et al., 2009. The isolation and characterization of a rhabdovirus from stone flounder, Kareius bicoloratus. Chin. J. Vet. Sci., 29: 277-282.
120. Sun YJ, Yue ZQ, Liu H, et al., 2010. Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR. J.Virol. Methods, 169: 391-396
121. Sun ZF, Hu CQ, Ren Ch, et al., 2006. Sensitive and rapid detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimps by loop-mediated isothermal amplification. Virol. Methods, 131(1): 41-46.
122. Tang ZM, Mei Q, Zhang CX, Zhu Y, Lu ZH, 2002. A comparative study of protein arrays immobilized on different substrates. In: Chinese Materials Research Society ed. Advanced Nanomaterials and Nanodevices. Presented at the 8th International Conference on Electronic Materials. Xi’an, China. p.359-369.
123. Tidona CA, Darai G, 1999. Lymphocystis disease virus (Iridoviridae), p. 908–911. In A. Granoff and R. G. Webster (ed.), Encyclopedia of virology. Academic Press, New York.
124. Tung CH, Gerszten RE, Jaffer FA, et al., 2002. A novel near-infrared fluorescence sensor for detection of thrombin activation in blood. Chembiochem, 3(2-3): 207-211.
125. Vamum SM, Woodbury RL, Zangar RC, 2004. A protein microarray ELISA for Screening biological fluids. Methods Mot. Biol., 278: 161-172
126. Victor NM, Tamara YM, 2006. Active bead-linked immunoassay on protein microarrays. Anal. Chim. Acta., 564: 40-52.
127. Walker R. 1962. Fine structure of lymphocystis disease virus in fish. Virology, 18:503-505.
128. Wang XJ, Zhan WB, 2006a. Development of an immunochromatographic test to detect White Spot Syndrome Virus of shrimp. Aquaculture, 255: 196-200.
129. Wang XJ, Zhan WB, Xing J., 2006b. Development of dot-immunogold filtration assay to detect white spot syndrome virus of shrimp. J. Virol. Methods, 132: 212-215.
130. Wang YC, Lo CF, Chang PS, Kou GH, 1998. White spot syndrome associated virus (WSSV) infection in cultured and wild decapods in Taiwan. Aquaculture, 164: 221-231.
131. Wang Z, Lee J, Cossins AR, et al., 2005. Microarray-based detection of protein binding and functionality by gold nanoparticle probes. Anal. Chem., 77(17): 5770-5774.
132. Wolf K, Gravell M, Malsberger RG, 1966. Lymphocystis virus: Isolation and propagation in centrarchid fish cell lines. Science, 151: 1004-1005.
133. Woodbury RL, Varnum SM, Zangar RC, et al., 2002. Elevated HGF levels in sera from breast cancer patients detected using a protein microarray ELISA. J. Proteome Research, 1(3): 233-237.
134. Xing J, Zhan WB, Zeng XH, Cheng SF, 2006. Detection of lymphocystis disease virus infection to flounder gill cells in vitro by monoclonal antibodies. High Technology Letters, 12(1): 103-108.
135. Xu RZ, Gan XX, Fang YM, et al., 2007. A simple, rapid, and sensitive integrated protein microarray for simultaneous detection of multiple antigens and antibodies of five human hepatitis viruses (HBV, HCV, HDV, HEV, and HGV). Anal. Biochem., 362: 69-75.
136. Yegneswaran S, Fernandez JA, Grifin JH, et al., 2002. Factor Va increases the affinity of factor Xa for prothrombin: a binding study using a novel photoactivable thiol-specific fluorescent probe. Chem. Biol., 9(4): 485-494.
137. Yu W, Gu N, Zhang H Q, et al. 2004. Microarray preparation based on oxidation ofagarose-gel. Sensors and Actuators B, 98: 83-91.
138. Zammatteo N, Jeanmart L, Hamels S, et al., 2000. Comparison between different strategies of covalent attachment of DNA to glass surfaces to build DNA microarrays. Anal. Biochem., 280(1): 143-50
139. Zhan WB, Wang YH, John LF, et al., 1998. White Spot Syndrome Virus Infection of Cultured Shrimp in China. J. Aqua. Animal Health., 10: 405-410.
140. Zhan W B, et al., 1999. Using developed monoclonal antibodies for detection of Penaeus chinensis WSSV infection. In: Proceeding of International Symposium on Progress and Prospect of Marine Biotechnology. Qingdao. 156-161.
141. Zhan WB, Li YQ, Sheng XZ, et al., 2010. Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China. Chinese Journal of Oceannology and Limnology. 28(6): 1214-1220.
142. Zhang QY, Ruan HM, Li ZQ, et al., 2003. Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines. Dis. Aquat. Org., 57: 27-34.
143. Zhou J, Larissa B, Pauline YH, et al., 2010. Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing. J. Immunol. Methods, 355: 40-51.
144. Zhu H, Snyder M, 2003. Protein chip tecimology. Curr. Opin. Chem. Biol., 7(1): 55-63.
145.陈信忠,龚艳清, 2006.鱼类病毒性神经坏死病研究进展.生物技术通报. S1: 141-146.
146.黄倢,宋晓玲,于佳等, 1995.杆状病毒的皮下及造血组织坏死-对虾爆发性流行病的病原和病理学.海洋水产研究, 161: 110-115.
147.黄倢,杨丛海,于佳等, 1995b. T-E染色法用于对虾暴发性流行病的现场快速诊断.海洋科学1: 29-34.
148.黄艳青,王桂堂,孙军等, 2003.黄颡鱼血清免疫球蛋白的纯化及分子量的初步测定.水生生物学报, 27(6): 654-656.
149.黄晓峰,张远强,张英起, 2004.荧光探针技术.北京:人民军医出版社, 404-408.
150.江凌晓,郭兆彪,陈泽良等, 2004.蛋白质芯片制作条件的优化.第一军医大学学报,24(11):1230-1232
151.姜红,李月红,吴东明,付云红, 2010.鱼类传染性造血器官坏死病临床诊断及检测.中国水产, 11:53-57.
152.江育林,李正秋, 1991.病毒感染的草鱼细胞产生类干扰素物质的研究.病毒学报, 7:30-35.
153.江育林, 1990.用酶联免疫吸附试验快速检测虹蹲的传染性胰脏坏死病病毒.水生生物学报, 14(3): 276-279.
154.江育林等, 1993,两种快速检测技术实验条件的探索及特性比较,第三次鱼病研讨会论文摘要汇编.
155.雷质文,史成银,莫照兰, 2002.人工感染白斑综合征病毒的克氏原螯虾的病理学试验.中国兽医科技, 32: 23-25.
156.李永芹.养殖鱼类病原菌检测芯片技术的建立. [博士学位论文].青岛:中国海洋大学水产学院, 2010.
157.李贵生,何建国,吴冰等, 2001.斑节对虾杆状病毒在感染对虾中肠腺中的分布.水产学报,25(2):141-147.
158.吕宏旭,汪岷,李红岩等, 2003.利用牙鲆鳃细胞系分离和培养淋巴囊肿病毒.青岛海洋大学学报, 33(2): 233-239.
159.刘志红,何为,韩旭等, 2007.琼脂糖凝胶蛋白芯片片基的制备及应用.分析化学, 35(5): 775-778.
160.刘荭,范万红,史秀杰等, 2006.国内养殖鱼类和进境鱼卵中传染性造血器官坏死病毒(IHNV)的检测及基因分析.华中农业大学学报. 25(5): 544-549.
161.黎小正,韦信贤,吴祥庆等, 2008.对虾Taura综合征病毒RT-PCR检测方法的改进及应用.中国预防兽医学报. 17(5): 525-529.
162.林天龙,陈强,俞伏松等, 2001.欧洲鳗血清免疫球蛋白纯化及部分特性分析.水产学报, 25(1): 52-57.
163.林炳承,秦建华, 2005.微流控芯片实验室.色谱. 23 (5): 456- 463.
164.李惠芳,刘荭,吕建强等, 2008. TaqMan实时荧光PCR快速检测斑点叉尾鮰病毒.长江大学学报,自然科学版. 5(1): 42-46.
165.马立人,蒋中华主编. 2002.生物芯片.第2版,北京:化学工业出版社. 40-56.
166.苗宏志,童裳亮,徐斌等, 2000.利用对虾原代细胞增殖对虾杆状病毒HHNBV的研究.生物工程学报. 16(2): 221-223.
167.曲径,沈海平,李笑刚等, 2001.威海地区养殖牙鲆鱼淋巴囊肿病流行病学调查.检验检疫科学, 11(6): 34-35.
168.孙广瑞,李小兵,王哲, 2007.制备分泌单克隆抗体杂交瘤细胞小鼠腹水的方法.中国现代医生. 45(5): 3-4.
169.绳秀珍.鱼类淋巴囊肿病毒的流行病学研究. [博士学位论文].青岛:中国海洋大学水产学院, 2006.
170.石锐,孙凯,王秀荣等, 2005.生物芯片在疾病诊断中的应用.畜牧兽医科技信息. 5: 7-8.
171.沈明山,罗文新,陈晋安等, 2000.厦门地区杂色花蛤的病毒观察与检测.海洋通报. 19(1): 93-96.
172.史成银,王印庚,黄倢等, 2005.大菱鲆红体病虹彩病毒ATPase基因的克隆与序列分析.高技术通讯. 5(15): 91-95.
173.孙修勤,张进兴, 1998.中国对虾肝胰腺的细小病毒病的直接荧光抗体诊断研究.黄渤海海洋. 16(4): 48-53.
174.王崇明,宋微波, 2002.栉孔扇贝一种球形病毒的分离纯化及其超微结构观察.水产学报. 26(2): 180-184.
175.王江勇,陈毕生,冯娟, 2000.杂色鲍裂壳病球状病毒的初步观察.热带海洋. 19(4): 82-85.
176.王炜,陈延,柯丽华等, 1990.草鱼出血病病毒武汉南湖株的精细结构与基因组及其多肽的研究.中国病毒学, 6(1): 41-49.
177.王艳,何为,刘志红, 2002.蛋白芯片的研究进展及其临床应用.国外医学:微生物学分册. 25(2): 7-9.
178.王艳霞,李宁,傅星等, 2003.原子力显微镜在生物力的测定应用.微电子技术. 7-8: 228~230。
179.许拉,黄倢,杨冰, 2008.病原检测基因芯片应用及在水产病害检测的前景.海洋水产研究. 29(1): 109-114.
180.谢树涛,何建国,杨晓明等, 2001.套式PCR检测斑节对虾白斑症病毒(WSSV).青岛海洋大学学报. 31(2): 220-224.
181.熊炜,邱璐,李健等, 2007. Real-time PCR方法和PCR方法检测虾白斑综合征病毒.中国预防兽医学报. 29 (2): 138-141.
182.颜桦.抗体芯片基本技术平台的建立及其在肝脏蛋白质组学研究中的应用。[硕士学位论文].陕西:西北大学, 2006.
183.曾伟伟,王庆,石存斌等, 2010.免疫学和分子生物学技术在水产动物疾病诊断中的应用.动物医学进展. 31(6): 111-117.
184.战文斌主编,水产动物病害学.青岛:中国农业出版社. 2004.
185.张奇亚,李正秋, 2001.胭脂鱼弹状病毒包涵体在培养细胞中的形成.中国兽医学报. 21(1): 37-40.
186.张永嘉,郭青,吴泽阳, 1997.云纹石板鱼淋巴囊肿病变过程的超微研究.海洋与湖沼. 28: 406-410.
187.朱建中,陆承平, 2001.对虾白斑综合征病毒在螯虾动物模型的感染特性.水产学报. 25: 47-58.
188.左小霞,高志贤,曹巧玲.葡萄球菌肠毒素A、B检测的免疫芯片技术研究.中国卫生检验杂志, 2008, 18(12): 2483-2488.