单抗免疫斑点法和组织印迹法检测侵染蝴蝶兰的建兰花叶病毒

应用抗建兰花叶病毒(CymMV)的单克隆抗体, 建立了快速检测蝴蝶兰病样的免疫斑点法(Dot-ELISA)和组织印迹法(Tissue blot-ELISA)。CymMV单抗稀释8000倍时, Dot-ELISA可检出病毒粗汁液的最大稀释度为1:10240; Tissue blot-ELISA中样品1次平切后1~5次印迹与Dot-ELISA样品1:80稀释结果相当, 6~8次印迹与Dot-ELISA 1:320稀释结果相当, 前8次印迹均可以得到满意的检测效果。Tissue blot-ELISA的灵敏度略低     

单抗免疫斑点法和组织印迹法检测百合无症病毒*

应用抗百合无症病毒(Lilysymptomlessvirus,LSV)的单克隆抗体,建立了快速检测田间样品的免疫斑点法(Dot-ELISA)和组织印迹法(Tissueblot-ELISA)体系。LSV单抗稀释2,000倍时,Dot-ELISA中病叶粗汁液可被检出的最大稀释度为1∶640。Tissueblot-ELISA中样品一次平切后第1次印迹与Dot-ELISA样品1∶40稀释的结果相当,前4次印迹均可获得满意的显色效果。常规Tissueblot-ELISA的灵敏度低于Dot-ELISA,     

植物病毒检测技术──组织印迹法

组织印迹法（Tissue blotting）是在酶联免疫吸附（Enzyme-Linked immunosorbent Assay ELISA）的基础上发展起来的植物病毒检测技术,该技术不仅保持了ＥＬＩＳＡ对病毒检测的灵敏度高,特异性强的特点,而且大大地简化了操作程序,对病毒的检测更加快速、简单、方便、印迹在硝酸纤维素膜上的样品能保存３个月以上,检测结果能直观地显示出病毒感染的部位。组织印迹技术尤其适用于植物病毒的大规模普查。     

阿特拉津高灵敏酶联免疫吸附检测法的建立

目的：建立高灵敏度的阿特拉津酶联免疫吸附检测法。方法：将间接竞争ELISA进行条件优化以提高检测灵敏度,包括包被抗原与一抗的最佳工作浓度筛选、选择一抗的最佳稀释度对包被抗原进行细化筛选、不同有机溶剂对竞争结合反应的影响、酶标二抗稀释度筛选等。用建立的酶联免疫检测法检测实际样品,再与高效液相色谱法（HPLC）检测进行比较。结果：利用优化后条件建立了阿特拉津间接竞争ELISA检测曲线,标准曲线的相关系数R²=0.9958,相关性较好。另由此标准曲线可得LOD （最低检出限）为1.972 ng/ml。用于检测实际样品,回收率在80%-120%之间。当添加样品浓度为（0~6） ng/ml时,该法的检测灵敏度高于HPLC。结论：新建立的阿特拉津ELISA特异性好、精密度高,可代替大型仪器用于阿特拉津实际样品检测。     

四种血清学方法检测烟草TMV、CMV的比较

本文应用ELISA－异种动物抗体双夹心法（DSM－ELISA）、ELISA－A蛋白酶联法（SPA－ELISA0、ELISA－斑点免疫法（Dot-ELISA)和葡萄球菌凝集法（SA－test)等四种血清学方法检测TMV、CMV感染的烟草病叶,结果以Dot-ELISA法灵敏度最高,其检测病叶粗汁液的稀释度为1：1280－2560,其次是DSM－ELISA和SPA－ELISA,均为1：640,SA－test灵敏度较低,为1：80。SPA－ELISA的非特异性反应比DSM－ELISA低,用健康汁液附抗血清,可以明显降低Dot-ELISA和SA-test的非特异性反应。     

小西葫芦黄花叶病毒外壳蛋白抗体制备

根据已报道的小西葫芦黄花叶病毒（ZYMV）序列设计特异引物,扩增ZYMV的全长外壳蛋白（CP）基因,插入原核表达载体pSBET后在大肠杆菌B121（DE3）plysS中诱导表达。通过12％SDS—PAGE和5％～20％梯度SDS—PAGE二次制备电泳纯化诱导产物,免疫小鼠,获得经过Western blot分析为特异的抗CP血清。硫酸铵沉淀法与ProteinA—Red Sepharose亲和层析相结合提取IgG,获得效价达1：4800的一抗,对西瓜和甜瓜田间样品的间接ELISA检测表明,ZYMV在田间普遍发生,研究制备的IgG可用于ZYMV检测。     

定量检测猕猴血清中重组人源化抗狂犬病毒单克隆抗体间接ELISA法的建立

目的：建立定量检测血清中重组人源化抗狂犬病毒单克隆抗体（HuMabs）NM57的间接ELISA法,为药代动力学研究提供一种简单快速的方法。方法：采用狂犬病毒糖蛋白包被酶标板、HRP标记的IgG-Fc段为标记抗体,建立定量检测HuMabsNM57的间接ELISA法,并对其特异性、灵敏度、精密度及准确度进行检测。结果：间接ELISA法检测HuMabsNM57的灵敏度为5ng／mL,组内及组间精密度分别为2．6％-6．0％、8．5％-11．3％。结论：建立了灵敏度高、特异性强的检测HuMabs NM57的间接ELISA法,精密度及准确度均符合药代动力学要求,可用于猕猴及人血清中HuMabsNM57的检测。     

仓储小麦中脱氧雪腐镰刀菌烯醇检测技术的优化

脱氧雪腐镰刀菌烯醇（Deoxynivalenol,DON）是小麦仓储过程中的重要危害因子，其污染水平是仓储公司定期检测的重要指标。为提高DON检测的平行性和准确度，文章对仓储小麦DON检测过程中样品制备和酶联免疫吸附法（ELISA）检测关键技术进行优化。结果表明：样品制备过程中，将扦样后的样品先粉碎再分样的处理方法（方法 2）明显优于先分样再粉碎的方法（方法 1），测定样品的相对标准偏差由25.28%降至7.42%。另外，将ELISA检测过程中两种不同的移液枪使用方法（前进移液法和反向移液法）进行比较，发现前进移液法在10次测定中结果相对标准偏差较小，平行性较好；且加样过程中，采用不贴壁加样方式会使检测值更加准确，平行性也较好。优化后的仓储小麦中DON毒素ELISA快速检测技术具有较好的平行性和准确度，可用于实际仓储小麦中DON毒素的准确、快速检测。     

重组甘蔗花叶病毒E株系外壳蛋白的纯化及其多克隆抗体制备

目的：制备可用于甘蔗花叶病毒（ScMV）E株系（ScMV-E）检测用多克隆抗体。方法：将ScMV-E外壳蛋白（CP）基因连接到pET29a（＋）上,经PCR检测、酶切及测序鉴定获得重组质粒pET29a-CP,在大肠杆菌BL21（DE3）中诱导表达重组ScMV-E外壳蛋白;采用His Trap Kit纯化目的蛋白,作为抗原免疫新西兰大白兔,制备特异性抗体;通过间接ELISA、Western blot和组织印迹法检测所制备抗体的特异性。结果：SDS-PAGE分析表明,重组融合蛋白含6个组氨酸标记,相对分子质量约43000;Western blot检测显示所获得的抗体特异性良好,间接ELISA法测得血清的效价为1：81 920;甘蔗叶片的组织印迹检测结果显示杂交效果良好。结论：制备的多克隆抗体可直接用于ScMV-E检测,并有望用于制备ScMV-E检测试剂盒。     

百合无症病毒单克隆抗体的制备及检测应用

用百合无症病毒(Lilysymptomlessvirus,LSV)免疫的BALBC鼠脾细胞与SP20鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗LSV单克隆抗体(MAb)的杂交瘤细胞(2A2、5H9、5H2和5E12),并分别制备它们的单抗腹水。4株单克隆抗体腹水间接ELISA效价达10-6,5H9和5E12的抗体类型及亚类均为IgG1,而2A2和5H2均为IgG3,4株单克隆抗体的轻链均为κ链。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)检测LSV的方法。病叶作1300倍稀释、提纯LSV病毒浓度为18ngmL(每孔的病毒绝对量为1.8ng)时,该方法仍能检测到病毒。利用ACP-ELISA检测了田间样品,发现LSV在百合上发病很普遍。     

A dot-ELISA mimicry western blot test for the detection of swine trichinellosis

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody affinity chromatography was developed for detecting Trichinella spiralis infection in swine. The test was as sensitive as an ELISA using excretory-secretory products as antigen and western blot analysis, and nearly as specific as the western blot. The dot-ELISA detected all of 20 low infections (0.08-4.74 larvae per gram of diaphragm), most of them by 5-6 wk postinfection. Sera from 1,960 farm-reared swine were tested by conventional ELISA, dot-ELISA, and western blot. Of the 1,960 sera, 262 (13.4%) were considered positive on conventional ELISA, 16 (0.82%) by dot-ELISA, and 15 (0.77%) by western blot. The improved specificity was achieved by employing species-specific denatured antigens. More importantly, the dot-ELISA was much simpler to perform than western blot analysis. The principles employed in this test can be adapted to other infectious diseases, such as AIDS.     

Cell wall proteins in white clover: influence of plant phosphate status

White clover (Trifolium repens L.) plants were grown in either P-containing liquid media, or in media with the sole source of phosphate removed (P-deprived). At 29 d, plants were harvested and a water-soluble whole tissue extract and an ionically-bound (1 M salt-extractable) cell wall protein extract made from root and leaf tissue. Acid phosphatase activity was highest in all extracts from root and leaf tissue excised from P-deprived plants, with the biggest difference (4.5-fold) in root cell walls. The smallest fold-increase was observed in the leaf water-soluble extract. The relative intensity of several, although not all, acid phosphatase isoenzymes was also highest in extracts from P-deprived plants. Up to the limits of detection used, no new acid phosphatase isoenzymes could be detected in extracts from plants maintained either in P-deprived or P-containing media. The complement of ionically-bound (1 M salt-extractable) cell wall glycoproteins in leaf and root tissue extracts maintained in P-deprived and P-containing media was also compared. Using SDS-PAGE and immuno-recognition with mAb 2.23, a monoclonal antibody that specifically recognises xylose/fucose mixed-type N-linked glycans, glycoproteins of 30 and 31 kDa were identified which were more prevalent in the P-deprived root cell wall. Further, a protein of 60 kDa was identified, which was prevalent in root cell wall extracts from plants maintained in P-containing media. The GNA lectin, which detects oligomannose N-linked structures, identified a glycoprotein of 37 kDa which was more prevalent in the P-deprived root cell wall.     

Differential expression of CMG peptide and crustacean hyperglycemic hormones (CHHs) in the eyestalk of the giant tiger prawn Penaeus monodon

Mouse antiserum against C-terminal amide of Pem-CMG (a peptide in the family of CHH/MIH/GIH) penta-deca peptide (RPRQRNQYRAALQRLamide=CMG-15) was generated and used for localization of the peptide in tissue and extract of the eyestalk of Penaeus monodon by means of immunohistochemistry and dot-ELISA in comparison with anti-T+ antiserum (T+=YANAVQTVamide : the putative C-terminal amide of crustacean hyperglycemic hormone (CHH) of Macrobrachium rosenbergii). The anti-CMG-15 antiserum did not show cross-reactivity to T+ peptide by dot-ELISA and vice versa for anti-T+ antiserum. In dot-ELISA of eyestalk extract of P. monodon after one step separation by RP-HPLC, anti-CMG-15 antiserum recognized different peptide fractions (F38-39) from those recognized by anti-T+ antiserum (F19, 40-41 and 47-51). Most of the T+ immunoreactive fractions (except F19) show higher hyperglycemic activity than the CMG immunoreactive fractions. In immunohistochemical localization, anti-CMG antiserum recognized only 2-3 neurons in medulla terminalis X-organ complex (MTXO) with long processes terminated in the sinus gland. The CMG-immunoreactive neurons were clearly distinct from CHH containing neurons situated in the same area. This evidence confirms the existing of CMG peptide which may play distinct roles from CHHs in hormonal regulation in P. monodon.     

Renalase's Expression and Distribution in Renal Tissue and Cells

To study renalase''s expression and distribution in renal tissues and cells, renalase coded DNA vaccine was constructed, and anti-renalase monoclonal antibodies were produced using DNA immunization and hybridoma technique, followed by further investigation with immunological testing and western blotting to detect the expression and distribution of renalase among the renal tissue and cells. Anti-renalase monoclonal antibodies were successfully prepared by using DNA immunization technique. Further studies with anti-renalase monoclonal antibody showed that renalase expressed in glomeruli, tubule, mesangial cells, podocytes, renal tubule epithelial cells and its cells supernatant. Renalase is wildly expressed in kidney, including glomeruli, tubule, mesangial cells, podocytes and tubule epithelial cells, and may be secreted by tubule epithelial cells primarily.     

Sieve element specific labeling with an anti-idiotypic monoclonal antibody mimic of the phytotoxin dothistromin

A monoclonal antibody, 12C9, an anti-idiotypic mimic of dothistromin, a toxin produced by Dothistroma pini, was found to label the cell wall of sieve elements in a number of different plant tissues and species. The antibody labeled apple leaf tissue, tobacco leaf mid vein, leaf and meristem, and Coprosma robusta leaf mid vein. Labeling was restricted to cell walls of sieve elements and did not label the companion cells or the lumen of the cells. The antibody labeled over a wide range of dilutions. This antibody could be used to differentiate sieve elements from other types of phloem. It could also be used to co-localize sieve elements and microorganisms such as phytoplasmas stained with DAPI.     

Sieve element specific labeling with an anti-idiotypic monoclonal antibody mimic of the phytotoxin dothistromin

A monoclonal antibody, 12C9, an anti-idiotypic mimic of dothistromin, a toxin produced by Dothistroma pini, was found to label the cell wall of sieve elements in a number of different plant tissues and species. The antibody labeled apple leaf tissue, tobacco leaf mid vein, leaf and meristem, and Coprosma robusta leaf mid vein. Labeling was restricted to cell walls of sieve elements and did not label the companion cells or the lumen of the cells. The antibody labeled over a wide range of dilutions. This antibody could be used to differentiate sieve elements from other types of phloem. It could also be used to co-localize sieve elements and microorganisms such as phytoplasmas stained with DAPI.     

Specific and Rapid Detection of Lily symptomless virus and Arabis mosaic virus in Lily by Dual IC‐RT‐PCR

Lily symptomless virus (LSV) and Arabis mosaic virus (ArMV) cause severe losses of quantity and quality of lily flower and bulb production. Specificity, sensitivity and speed of detection methods for viruses need to be improved greatly to prevent LSV and ArMV from spreading from infected lilies. A dual IC‐RT‐PCR procedure for detection was developed in which the antibodies of LSV and ArMV were mixed and the mixture used to coat the PCR tubes. The particles of the two viruses were captured by the respective antibodies. Interference by other RNA viruses in infected lily was eliminated in the RT‐PCR. Also, an RNA extraction step was omitted. The dual IC‐RT‐PCR products of LSV and ArMV were 521 bp and 691 bp, respectively. The specificity of the method was validated; only LSV and ArMV of four viruses were detected by dual IC‐RT‐PCR. The sensitivity of the detection method is 1 mg leaf tissue and higher than DAS‐ELISA due to enrichment by dual immunocapture.