Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

Tracking fungi in soil with monoclonal antibodies

Species of the genus Trichoderma are ubiquitous soil-borne fungi that exhibit antagonism towards a number of economically important plant-pathogenic fungi and oomycetes. This review discusses recent developments in the use of monoclonal antibodies to detect these fungi in their natural soil environments and to quantify their population dynamics during antagonistic interactions with saprotrophic competitors in soil-based systems. Immunological approaches to detection and quantification are examined in relation to conventional plate enrichment techniques and to nucleic acid-based procedures. An example of recent research using a mAb-based assay to quantify the effects of saprotrophic competition on the growth of Trichoderma isolates in mixed species, soil-based, microcosms is presented. Future technological developments in immunoassays for tracking Trichoderma populations in soil are discussed and results presented showing the accurate detection and visualization of a plant growth-promoting isolate of T. hamatum in the rhizosphere of lettuce using mAb-based immunodiagnostic assays.     

Characteristics of monoclonal antibodies against Piscirickettsia salmonis

A panel of 28 monoclonal antibodies against Piscirickettsia salmonis was produced using a purified fraction of the bacterium. To determine their specificity to the pathogen, the antibodies were assayed by ELISA and indirect immunofluorescence microscopy. Six monoclonal antibodies were selected based on their strong reaction against P. salmonis and absence of cross-reactivity with other common fish pathogens. Western blot analysis showed that the antibodies reacted to several antigens of P. salmonis . Immunofluorescence assays revealed that these antibodies reacted with the same specificity to different isolates of P. salmonis obtained from the south of Chile. This panel of monoclonal antibodies represents an important tool to develop simple, rapid, sensitive and highly specific methods for the detection of the pathogen and diagnosis of the disease.     

Presence of anti-Neoparamoeba sp. antibodies in Tasmanian cultured Atlantic salmon, Salmo salar L

Previous studies have indicated that when Atlantic salmon, Salmo salar L., are exposed to Neoparamoeba sp. the fish produce anti-Neoparamoeba sp. antibodies. It appears unlikely that these antibodies elicit any specific protection against amoebic gill disease (AGD) as fish with demonstrable activities have been affected by AGD. Experiments were conducted on Atlantic salmon cultured throughout Tasmania to assess the natural production of antibodies towards Neoparamoeba sp. Fish were sampled from areas where AGD was prevalent and from areas where there had been no reported cases. An enzyme-linked immunosorbent assay (ELISA) was used to measure anti-Neoparamoeba sp. antibody activities in serum. All fish from sea water had antibody activities greater than the negative control fish, including fish from areas with no reported cases of AGD. Time trial samples indicated that time after transfer to sea water did not appear to be a significant (P > 0.05) factor in antibody activity, however location was (P < 0.05). There was no agreement (corrected kappa value, 0.16) between the ELISA result and the isolation of Neoparamoeba sp. from the gills of the same fish. The results suggest that Atlantic salmon in seawater culture in Tasmania produce anti-Neoparamoeba sp. antibodies regardless of infection history, suggesting the presence of Neoparamoeba sp. in the environment.     

猕猴桃种传潜隐病毒外壳蛋白的原核表达及多克隆抗体制备

猕猴桃种传潜隐病毒(actinidia seed borne latent virus, ASbLV),属于乙型线状病毒科Betaflexiviridae李属病毒属Prunevirus,是一种在我国猕猴桃上广泛发生的病毒。本研究通过RT-PCR方法克隆ASbLV的外壳蛋白基因,并连接到原核表达载体pET28a(+)上,转化大肠杆菌Rosetta (DE3),经IPTG诱导后可表达分子量约为28 kD的融合蛋白。经过Ni²⁺-NTA树脂纯化融合蛋白,然后以其为抗原制备多克隆抗体。Western blot结果表明,多克隆抗体的效价为1∶4 000。该多克隆抗体只与ASbLV发生特异性反应,而不与猕猴桃病毒1、猕猴桃病毒A、苹果茎沟病毒、柑橘叶斑驳病毒、黄瓜花叶病毒和马铃薯X病毒发生反应,说明该多克隆抗体特异性良好。利用间接ELISA对36份猕猴桃田间样品进行了ASbLV检测,检测结果表明其中20份样品被ASbLV侵染,且间接ELISA检测结果与RT-PCR检测结果一致。本研究建立的间接ELISA方法能够有效地应用于猕猴桃田间样品中的ASbLV检测。     

三唑磷酶联免疫吸附测定法中包被抗体稳定剂的研究

选择了甘油、山梨醇、聚乙二醇(PEG)、卵清蛋白(OVA)、多肽、糖、氨基酸等试剂,通过经验法和正交法相结合的手段配制了一系列稳定剂,对吸附在酶标板上的三唑磷多克隆抗体进行处理(37℃,1 h)后,再在37℃下连续贮存 7 d,利用直接竞争ELISA法对不同稳定剂处理的包被抗体免疫活性、亲合性及检测灵敏度进行检测,并与未经稳定剂处理的对照进行比较,筛选得到效果较好的稳定剂 1 (质量分数:甘油2.5%,氨基酸1.5%,蛋白胨3.0%,离子螯合剂0.1%,防腐剂0.01%)。用稳定剂 1 处理包被抗体后,4~6℃下保存半年及37℃下保存14 d的试验结果表明,抗体的活性相对保持率分别为97.8%和94.2%;其免疫活性、亲合性(I50分别为68.43和54.38 ng/mL)及灵敏度(I10分别为3.72和 3.22 ng/mL)与常规方法包被的抗体(包被好后不贮存,直接检测,I50为60.73 ng/mL,I10为 3.11 ng/mL)无明显差异;冻融试验表明,经稳定剂 1 处理的三唑磷抗体在反复冻-融次数不超过8次时其活性也是稳定的。说明筛选出的稳定剂可以显著提高三唑磷多克隆包被抗体的稳定性,可用于三唑磷ELISA试剂盒的生产。     

山羊痘病毒糖蛋白基因ORF112的原核表达及多克隆抗体的制备

为探索山羊痘病毒(GTPV)糖蛋白基因ORF112在疫苗和诊断中的应用,应用PCR技术扩增GTPV弱毒疫苗株AV 41ORF112基因,将其克隆到pET-32a载体,转化感受态细胞BL21,经IPTG诱导后获得与预期大小相符的约37ku的融合蛋白,为可溶性和包涵体表达。应用镍离子亲和树脂对可溶性表达的目的蛋白进行纯化,然后用纯化的融合蛋白免疫Balb/c小鼠,制备多克隆抗体。免疫荧光试验表明该多克隆抗体可以与GTPV反应,为GTPV新型疫苗和诊断试剂的研究奠定了基础。     

抗ICAM-1单克隆抗体的生物学功能研究

本研究通过体外细胞试验和体内动物试验,对已制备的抗细胞间粘附分子1(ICAM-1)单克隆抗体(MAb)4F1和2C5的生物学活性进行检测.间接免疫荧光试验表明:这两株ICAM-1 MAb可以与人血管内皮细胞ECV304细胞中的ICAM-1结合.以乳酸脱氢酶释放法检测ICAM-1 MAb抑制单核细胞与血管内皮细胞的黏附作用.4F1和2C5 MAb分别可以使细胞黏附降低34.4％和26.9％.二甲苯致小鼠耳廓肿胀试验和毛细血管通透性试验表明:ICAM-1 MAb能够抑制小鼠耳廓肿胀度,使伊文思蓝渗出量降低.本研究证明所制备的MAb均具有阻断ICAM-1及抑制炎症反应的功能,其中4F1生物学活性高于2C5.     

非洲猪瘟病原学及单克隆抗体制备技术研究进展

非洲猪瘟是由非洲猪瘟病毒引起猪的高度接触性、传染性、出血性以及高死亡率的传染病。20世纪中期以来,已在非洲、欧洲和美洲等数十个国家流行,并在近几年内蔓延至欧亚两洲接壤处的格鲁吉亚、亚美尼亚、阿塞拜疆以及俄罗斯境内,其一旦侵入我国,将会给我国养猪业带来极大的危害。非洲猪瘟病原学研究以及制备相应的单克隆抗体对非洲猪瘟病毒快速诊断技术研究和疫苗研制有着重大的现实意义。主要从病原学和单克隆抗体制备方面对非洲猪瘟的研究进展进行了综述。     

甲型H1N1流感病毒NS1蛋白核仁定位的研究

为研究2009年甲型H1N1流感病毒的NS1蛋白的核仁定位情况,采用RT-PCR对其NS1基因进行了扩增,将其克隆至PEGX-KG载体,构建重组质粒KG-NS1,转化大肠杆菌BL21,IPTG诱导表达重组蛋白.然后采用GST柱亲和层析方法纯化NS1重组蛋白,免疫家兔来制备多抗,Western blot检测抗体.通过间接免疫荧光对表达不同长度NS1 (NS1-219、NS1-230、NS1-237)的3种重组流感病毒进行了核仁定位的研究,3种重组毒的NS1蛋白存在于细胞核和细胞质,但都不能定位于核仁,说明NS1蛋白的截短与否并不影响其核仁定位,其生物学意义有待于进一步研究.