应用单克隆抗体的免疫组织化学法研究雏鸭体内鸭瘟病毒的分布

本实验应用了免疫组织化学的单克隆抗体间接酶标染色法，对人工感染鸭瘟病毒雏鸭的组织切片进行染色观察。旨在研究病毒在鸭体内分布，对其进行定位。研究结果显示，鸭的心脏、肝脏、脾、胸腺、肠、法氏囊、胰、肺、肾等组织的细胞浆内均出现了染色的特异阳性反应物。结果表明，鸭瘟病毒广泛分布于感染雏鸭的各种组织器官，并造成一定的组织病理变化。     

磺胺二甲嘧啶单克隆抗体的制备及特性鉴定

采用重氮化法合成磺胺二甲嘧啶(SM_2)-人血清白蛋白(HSA)免疫抗原和SM_2~-卵清白蛋白(OVA)包被抗原。经紫外光谱扫描法确认SM_2与载体蛋白偶联成功；经计算SM_2与HSA、OVA的结合比分别为9：1和15：1。利用杂交瘤技术和有限稀释法经过5次亚克隆,得到三株特异性稳定分泌SM_2抗体的杂交瘤细胞,经鉴定该单克隆抗体免疫球蛋白亚类为IgG_1,为入链,分子量为162Ku,染色体数目90条左右,亲和常数为6．1×10~(12)M~(-1)。与其他四种磺胺药和两种载体蛋白HSA、OVA均无交叉反应。     

副猪嗜血杆菌间接ELISA抗体检测方法的建立

采用福尔马林灭活的副猪嗜血杆菌全菌体作为包被抗原,建立了检测副猪嗜血杆菌抗体的间接ELISA方法。经试验确定副猪嗜血杆菌全菌体的包被浓度为2·24×107CFU/孔、检测血清为1∶200稀释,同时确立了间接ELISA的最佳反应条件。该方法有很高的特异性和重复性,14个发病猪场100份血清检测结果Hps抗体阳性检出率为94%,明显高于细菌分离鉴定检测结果。     

规模化猪场脑心肌炎病毒感染的血清学调查

脑心肌炎病毒(EMCV)可引起猪的脑炎、心肌炎和母猪繁殖障碍,该病已在世界上多个国家暴发和流行。我国对EMCV及其所致疾病的研究工作还处于起步阶段,为了揭示EMCV在我国规模化猪场的感染情况,我们应用酶联免疫吸附试验(ELISA)对2005-2006年间采集自全国13个省市46个规模化猪场的3250份血清样本进行了EMCV抗体检测,结果显示,各省阳性率在39.64%~90%之间,平均抗体阳性率为72%,监测的46个猪场都存在EMCV感染,猪场感染阳性率为100%;对不同生长阶段猪群检测结果表明,EMCV抗体阳性率随猪龄的增长而升高,且不同生长阶段猪抗体阳性率差异极显著;成年公猪的抗体阳性率高于成年母猪。本项调查表明,我国规模化猪场已经普遍感染EMCV,这应引起我国养猪业警惕,并做好该病的综合防控。     

禽流感血清抗体与卵黄抗体消长规律比较研究

应用禽流感病毒二价（H5、H9）油乳剂灭活疫苗免疫160日龄非免疫蛋鸡，免疫接种后分别于第0、7、14、21、28、35 d采集相应的鸡蛋和血清，采用血凝抑制(HI)试验监测血清及卵黄中H5、H9抗体的消长规律，结果表明：H5、H9血清抗体于免疫后7 d时开始产生，14 d时到中等水平，21 d时达到最高值，一直维持到35 d之后；而H5、H9卵黄抗体与血清抗体水平相比相对滞后7 d左右；卵黄抗体与血清抗体在28 d时均达到高峰值并一直维持到35 d之后，28 d后血清和卵黄抗体达到相同水平。本研究为临床上以禽流感卵黄抗体检测替代血清抗体检测及以后禽流感高免卵黄抗体的研究提供了依据和数据。     

抗H5亚型禽流感病毒血凝素蛋白特异性单克隆抗体的研制及鉴定

以抗H5亚型禽流感病毒（AIV）JSD株鸡胚尿囊液免疫8周龄BALB/c小鼠,第4次免疫后取其脾淋巴细胞与SP2/0-Ag-14骨髓瘤细胞融合,用血凝抑制试验（HI）对杂交瘤进行筛选,经3次亚克隆后,共获得4株针对血凝素（HA）蛋白的特异性单克隆抗体,分别命名为：2D2、2C8、3D3和3D11。该4株单抗的小鼠腹水的HI效价在2^13～2^15,ELISA效价为1.2×10^5～5.1×10^5。亚类鉴定结果表明,2D2和3D11属于IgG1,2C8属于IgG3,3D3属于IgG2a亚类;特异性试验结果表明,上述4株单抗仅与H5 AIV毒株发生特异性反应,而不与其他亚型AIV以及NDV、IBV和EDSV-76等病毒反应;鸡胚中和试验结果显示,4株单抗均具有较好的中和活性。本研究成功获得4株针对H5 AIVHA蛋白的特异性单抗,为临床H5 AIV的血清学监测及鉴定提供了必需的试剂。     

抗猪附红细胞体单克隆抗体的制备

以纯化的猪附红细胞体免疫BALB/c小鼠,运用淋巴细胞杂交瘤技术进行细胞融合,并用间接ELISA方法进行筛选,经过间接ELISA方法、免疫印迹和间接免疫荧光试验进行鉴定,共获得5株分泌抗猪附红细胞体单克隆抗体的杂交瘤细胞,分别命名为1H1、1H2、3A5、5B1和7E11,其单抗亚类鉴定分别属于IgG2b、IgG1、IgG2b、IgG2b和IgG2b。这5株McAb均能与猪附红细胞体全菌蛋白发生特异性反应,而不与猪肺炎支原体、猪链球菌、大肠杆菌和猪繁殖与呼吸综合征病毒发生反应,并且1H1、3A5和5B1能识别同一抗原位点,1H2和7E11识别另一抗原位点。     

H5N1亚型禽流感灭活疫苗免疫鸵鸟后抗体消长规律及免疫程序的研究

为了探讨鸵鸟对H5亚型禽流感灭活疫苗的免疫原性,做好鸵鸟禽流感的防控工作,采用哈尔滨兽医研究所研制的重组H5N1亚型禽流感疫苗,不同剂量免疫鸵鸟,用HI方法检测母源抗体和免疫抗体,根据母源抗体的衰减和免疫抗体的消长规律确定首免和再免日龄.结果表明,雏鸵鸟母源抗体能维持约3周～8周;8周龄时分组首免,C1组产生的免疫反应优于C2组,抗体峰值能达到7.50 log2,维持时间为9周左右;17周龄时C1组以3.0 mL/羽进行二免,2周后抗体达到7.40log2,3周～7周抗体维持在高峰值,最高达8.80log2,以后逐渐下降,有效抗体水平能维持至接种后25周左右.二免后25周进行三免,以5 mL/羽三免后2周抗体可达到9.80log2,2周～4周抗体维持在最高峰,以后缓慢下降,期间抗体时有起伏,但有效抗体水平约可维持1年时间.三免后45周内抗体水平合格率均在70%以上.根据抗体消长规律,初步推荐了鸵鸟的免疫程序.     

Thrombolytic properties of a conjugate of liposomal urokinase with a monoclonal antibody specific for cross-linked fibrin in the model of rabbit artery thrombosis

AIM: To observe the targeting thrombolytic effect of a monoclonal antibody specific for cross-linked fibrin connected with liposomal urokinase（UK） in the model of rabbit artery thrombosis.METHODS: Preparation of thrombolytic solutions: with the method of controllable dialysis eradicator in liposomat,empty liposomes,liposomally entrapped urokinase and liposomally entrapped urokinase linked with D-dimer antibody were made.Experiment in vivo: a rabbit thrombosis model of abdominal aorta was induced by ferric chloride.When the blood pressure fall to the lowest,5 different solutions were separately imported (PBS,maximal-level UK,Ab/Lip/UK,Ab/UK-Lip,Ab-UK-Lip) and observed for 40 min continuously.RESULTS: The varieties of 5 groups blood pressures were analyzed with q-test.Significant differences were observed among Ab-UK-Lip group,maximal-level UK group and others (P<0.01).There were marked differences between Ab-UK-Lip group and others in wet weight of thrombuses (P<0.05).However,the quantity of UK in Ab-UK-Lip group was 1/3 of that in maximal-level of UK group.CONCLUSION: The thrombolytic effect on Ab-UK-Lip group is similar to that on maximal-level of UK group,but the dosage of UK is less,and the side-effect is light.This shows,as a homing-device of liposomal urokinase,the D-dimer antibody has targeting effect and the agent of Ab-UK-Lip will be attached importance by-and-by.     

Effect of different PRA serum levels from patients with β-thalassemia on the proliferation and differentiation potential of the hematopoietic stem cells /progenitor cells of cord blood

AIM: To probe the effect of different panel reactive antibody(PRA) serum levels from patients with β-thalassemia on the proliferation and differentiation potential of the hematopoietic stem /progenitor cell of cord blood.METHODS: 1×105 mononuclear cells (MNCs) isolated from umbilical cord blood were incubated with different PRA serum levels (0 μL,50 μL,100 μL) respectively and complement,inoculated into the methylcellulose cultural system.The proliferation and differentiation potential of the hematopoietic stem /progenitor cell of cord blood by the colony formation assay were detected on day 7 and day 14,respectively.RESULTS: After culture of 7 days,the total colonies and CFU-GM were 88.20±9.41,79.00±11.39 in group A and 88.60±9.12,79.20±10.44 in group B,which were significantly higher than those of 20.60±7.39,15.20±4.66 in group C and those of 4.00±2.05,1.40±0.51 in group D (P<0.01).Meanwhile compared with group A and group E,no significant difference was found (P>0.05).After culture for 14 days,the total colonies and CFU-GM were 216.00±31.10,117.40±24.80 in group A and 213.20±31.06,116.00±19.75 in group B,which were significantly higher than those of 97.80±14.43,32.80±8.10 in group C and those of 31.40±13.41,8.40±4.30 in group D (P<0.01).The CFU-GEMMs were 45.60±8.51 in group A and 42.60±7.03 in group B,which were significantly higher than those of 20.80±6.96 in group C and those of 7.80±6.06 in group D (P<0.05).The BFU-MK was 12.80±4.42 in group A and 11.00±2.74 in group B respectively,which were significantly higher than that of 1.00±0.55 in group D (P<0.05).The CFU-E in group B 17.20±4.03 was significantly higher than that of 5.60±2.87 in group D (P<0.05).Meanwhile compared with group A and group E,no significant difference was found (P>0.05).By the Kendall test,there were negative correlations between the level of PRA serum and the total colonies,CFU-GM on day 7,the total colonies,CFU-GM,CFU-GEMM,BFU-E,BFU-MK on day 14 (tau-b=-0.793,-0.849,-0.808,-0.804,-0.645,-0.674,-0.624,P<0.01).There was a negative correlation between the level of PRA serum and CFU-MK on day 14 (tau-b=-0.466，P<0.05).CONCLUSION: PRA sera inhibit the colony in the colony cultures of the hematopoietic stem cells/progenitor cells in cord blood.The inhibition depends on the level of PRA sera.The higher the level of PRA sera,the stronger the inhibition is observed in our study.