检测莱克多巴胺胶体金免疫层析试验的建立

采用混合酸酐法制备莱克多巴胺-OVA偶联抗原,胶体金标记莱克多巴胺单克隆抗体,建立了检测莱克多巴胺的胶体金免疫层析试验。该试验具有较好的敏感性和特异性,最低可检测10ng/mL的莱克多巴胺,而与克伦特罗和沙丁醇胺无交叉反应。胶体金免疫层析试验对尿液和饲料样品中莱克多巴胺的最低检测量分别为10ng/mL和0.01mg/kg。     

莱克多巴胺多克隆抗体的制备

采用多元酸酐法活化盐酸莱克多巴胺，用混合酸酐法将活化的莱克多巴胺与载体蛋白（KLH，BSA）偶联，制备了莱克多巴胺免疫原KLH-Rac和包被抗原BSA-Rac。以KLH-Rac免疫新西兰白兔获得了莱克多巴胺多克隆抗体，以BSA-Rac为包被抗原，采用间接ELISA检测抗血清，其效价达到1：6000，间接竞争ELISA测得抗血清的IC50值约为5ng／mL，其与克伦特罗、沙丁胺醇、特布它林的交叉反应性小于0．01％。     

莱克多巴胺人工抗原的合成与鉴定

用戊二酸酐将盐酸莱克多巴胺活化成莱克多巴胺-戊二酸酐半醛,用混合酸酐法将莱克多巴胺-戊二酸酐半醛与KLH和BSA偶联,合成免疫原和包被抗原,经核磁共振法和紫外吸收法鉴定,偶联成功.用紫外吸收法测定免疫原和包被抗原的偶联比分别为24：1和2.5：1.用免疫原免疫新西兰白兔,采用间接ELISA法检测抗体效价,采用间接竞争ELISA法检测IC50值,获得了效价为1：6 000以上、IC50值为10 ng/mL的多克隆抗体,证明合成的人工免疫原具有免疫原性.     

进出口食用动物、饲料中盐酸克伦特罗ELISA检测方法的建立

为了建立进出口食用动物、饲料中盐酸克伦特罗残留快速检测技术,试验建立间接竞争酶联免疫吸附试验(ELISA)方法进行检测。结果表明:包被抗原的最适质量浓度为20.0μg/mL,抗体的最佳稀释度为1∶5 000,半抑制浓度(IC50)=2.1 ng/mL;该方法的最低检测限达0.05 ng/mL,与沙丁胺醇和莱克多巴胺的交叉反应率分别为8.28%、7.75%,平均加标回收率为94.52%。该方法灵敏度高,样品前处理简便、快速,适用于进出口食用动物、饲料中盐酸克伦特罗残留的大批量现场检测。     

苯乙醇胺A单克隆抗体的研制及ELISA检测方法的建立

为制备苯乙醇胺A(phenylethanolamine A,PA)单克隆抗体,建立一种针对苯乙醇胺A的快速、简便、灵敏度高的检测方法,本试验采用重氮化法将制备的苯乙醇胺A衍生物分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)进行偶联作为免疫原和包被原。利用弗氏佐剂充分乳化免疫原(PA-BSA),按常规免疫程序免疫6～8周龄的雌性BALB/c小鼠,选择血清抗体效价较高的小鼠,取其脾细胞与SP2/0骨髓瘤细胞以PEG法进行细胞融合。结果显示,经3次细胞亚克隆筛选后,最终筛选出一株可稳定分泌抗苯乙醇胺A单克隆抗体的杂交瘤细胞株,命名为D6H8,利用此细胞以小鼠体内诱生法制备抗体。经鉴定,D6H8腹水抗体亚型为IgG1,轻链为κ链;纯化后的抗体与沙丁胺醇、盐酸克伦特罗、盐酸异丙肾上腺素和盐酸去氧肾上腺素均无明显的交叉反应(CR<0.18%),表明该抗体特异性良好。利用此抗体建立针对苯乙醇胺A药物残留检测的间接竞争ELISA方法,结果表明,腹水抗体效价为1∶12 800,猪肉中苯乙醇胺A添加浓度在5～1 000 ng/mL时线性关系良好,标准工作曲线为y=0.3861x-0.1845 (R^2=0.990),其IC₅₀为58.88 ng/mL,LOD为3.83 ng/mL,回收率在85.96%～104.32%之间。综上所述,本试验成功建立了检测苯乙醇胺A药物残留的间接竞争ELISA方法,该方法灵敏度高、稳定性较好。     

苯乙醇胺A单克隆抗体的研制及ELISA检测方法的建立

为制备苯乙醇胺A(phenylethanolamine A,PA)单克隆抗体,建立一种针对苯乙醇胺A的快速、简便、灵敏度高的检测方法,本试验采用重氮化法将制备的苯乙醇胺A衍生物分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)进行偶联作为免疫原和包被原。利用弗氏佐剂充分乳化免疫原(PA-BSA),按常规免疫程序免疫6～8周龄的雌性BALB/c小鼠,选择血清抗体效价较高的小鼠,取其脾细胞与SP2/0骨髓瘤细胞以PEG法进行细胞融合。结果显示,经3次细胞亚克隆筛选后,最终筛选出一株可稳定分泌抗苯乙醇胺A单克隆抗体的杂交瘤细胞株,命名为D6H8,利用此细胞以小鼠体内诱生法制备抗体。经鉴定,D6H8腹水抗体亚型为IgG1,轻链为κ链;纯化后的抗体与沙丁胺醇、盐酸克伦特罗、盐酸异丙肾上腺素和盐酸去氧肾上腺素均无明显的交叉反应(CR0.18%),表明该抗体特异性良好。利用此抗体建立针对苯乙醇胺A药物残留检测的间接竞争ELISA方法,结果表明,腹水抗体效价为1∶12 800,猪肉中苯乙醇胺A添加浓度在5～1 000 ng/mL时线性关系良好,标准工作曲线为y=0.3861x-0.1845 (R~2=0.990),其IC_(50)为58.88 ng/mL,LOD为3.83 ng/mL,回收率在85.96%～104.32%之间。综上所述,本试验成功建立了检测苯乙醇胺A药物残留的间接竞争ELISA方法,该方法灵敏度高、稳定性较好。     

酶联免疫吸附法检测猪肉和猪尿中莱克多巴胺残留

以莱克多巴胺单克隆抗体间接竞争酶联免疫吸附（ELISA）法测定猪肉和猪尿中莱克多巴胺残留。该法对莱克多巴胺的检测限为0.8ng/mL（g）,在空白组织中的添加回收率为80.4%～109.5%,变异系数为5.8%～12.6%。     

气相色谱-质谱法同时测定猪肝中盐酸克伦特罗、莱克多巴胺残留

猪肝用乙酸乙酯、盐酸溶液提取后，经SCX固相萃取柱净化、BSTFA衍生化，定容后用气相色谱-质谱（GC／MS）外标法测定。测定结果表明：采用该方法，猪肝中盐酸克伦特罗和莱克多巴胺的检出限分别为0．3、1．0ng／g。盐酸克伦特罗在3．0～300ng／mL范围内与峰面积具有良好的线性关系，平均回收率为85．6％；莱克多巴胺在10～1000ng／mL范围内与峰面积具有良好的线性关系，平均回收率为76．6％。该方法简便可靠、干扰小、灵敏度高，可用作猪肝中克伦特罗和莱克多巴胺残留的同时测定。     

盐酸四环素人工抗原的合成及鉴定

本试验旨在合成盐酸四环素人工抗原,为其单克隆抗体的制备奠定试验基础。将盐酸四环素(TCH)发生霍夫曼降解和重氮化反应生成重氮盐,再与蛋白质载体(BSA,OVA)偶联生成免疫原和包被原,对偶联产物采用三氯化铁及紫外扫描法鉴定偶联成功,且免疫原和包被原的摩尔结合比分别约为3.4∶1和4.2∶1。用免疫原免疫BALB/c小鼠,用间接ELISA检测抗体效价,采用竞争ELISA法测IC50,获得了效价为1∶51200I、C50为16.91 ng/mL的多克隆抗体,最终证明人工抗原制备成功。     

不同品牌“瘦肉精”检测卡质量验证

对通过农业部备案的A、B、C 3个生产厂家的盐酸克伦特罗、沙丁胺醇、莱克多巴胺快速检测卡进行质量验证。结果表明,3家的产品对盐酸克伦特罗的准确率检测最高,检测阈值符合产品要求,莱克多巴胺和沙丁胺醇的准确率低,其中沙丁胺醇的检出限均不符合产品说明的限量标准。     

鱼腥草多糖对MA104和MARC145细胞活力影响的比较研究

研究鱼腥草多糖(houttuynia cordata polysaccharide,HCP)对MA104和MARC145细胞活力的影响,筛选梯度浓度的鱼腥草多糖对MA104和MARC145细胞促生长最佳浓度剂量范围和毒性剂量.通过水提醇沉法提取HCP,得糖率为8.18％;分别采用MTT法和细胞平板计数法绘制MA104和...     

Effects of cidofovir on cell death and replication of feline herpesvirus-1 in cultured feline corneal epithelial cells

OBJECTIVE: To assess the effect of cidofovir on viability of feline corneal epithelial (FCE) cells, replication of feline herpesvirus (FHV)-1, and virus-induced cytopathic changes. SAMPLE POPULATION: Healthy eyes from 14 recently euthanatized cats. PROCEDURE: Cidofovir at concentrations ranging from 0.05 to 0.000005 mg/mL was added to primary cultures of FCE cells, and cytopathic changes and effects on cell proliferation and cell viability were determined during the subsequent 48 hours. Efficacy of cidofovir (0.02 and 0.05 mg/mL) to prevent in vitro infection of FCE cells with FHV-1 was determined during 72 hours of culture by assessing viral cytopathic effects and viral titers. RESULTS: Cidofovir at concentrations of 0.05, 0.005, and 0.0005 mg/mL significantly reduced mean viable cell counts, and cidofovir at a concentration of 0.05 mg/mL significantly reduced the percentage viability of cultured FCE cells. Minimal cytopathic changes were observed at concentrations of 0.02 and 0.05 mg of cidofovir/mL. Cidofovir at concentrations of 0.05 and 0.02 mg/mL abrogated the cytopathic effects attributable to FHV-1 infection and reduced viral titers from > or =10(14) TCID(50)/mL to < or =10(3.5) TCID50/mL. CONCLUSIONS AND CLINICAL RELEVANCE: Cidofovir in vitro was highly efficacious against FHV-1 infection of a primary culture of FCE cells but had cytostatic effects on cultured cells.     

泰氏锥虫抗原制备及实验室诊断研究

应用体外培养的泰氏锥虫制备可溶性抗原Ⅰ、抗原Ⅱ和代谢抗原.经测定,其蛋白含量每毫升分别为6.5mg、7.4mg和7.1mg.薄层等电聚焦电泳测定结果表明,抗原Ⅰ出现22条区带,抗原Ⅱ21条区带,代谢抗原28条区带,对照的伊氏锥虫琼脂免疫扩散抗原14条区带.经分析,泰氏锥虫抗原和伊氏锥虫抗原有4条区带在同一迁移率上.琼脂免疫扩散反应中,3种泰氏锥虫抗原均与相应免疫兔血清发生沉淀反应,抗原Ⅰ出现1条致密沉淀线,抗原Ⅱ和代谢抗原出现2～3条沉淀线,抗原效价为1:4～16.免疫电泳显示了类似的结果,抗原Ⅰ与免疫兔血清出现1条弧形沉淀线,抗原Ⅱ和代谢抗原与免疫兔血清出现了3条弧形沉淀线.间接血凝试验结果表明,泰氏锥虫自然感染牛血清效价为1:20～40,免疫兔血清为1:1280～5120.所制泰氏锥虫抗原对伊氏锥虫和媾疫锥虫血清也能很好地发生交叉反应,3份伊氏锥虫病马血清和3份伊氏锥虫人工感染兔血清血凝效价分别为1:10～40和1:8～1024;5份媾疫马血清有4份血凝效价为1:20～320.4份环形泰勒虫病牛血清均为阴性.     

Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages

The effect of ovotransferrin (ovoTF), human lactoferrin (hLF) and bovine lactoferrin (bLF) on the obligate intracellular pathogen Chlamydophila (Cp.) psittaci was evaluated using a model of Buffalo Green Monkey kidney (BGM) cells and HD11 chicken macrophages as artificial hosts. Firstly, the effect of transferrins on the infectivity of the bacteria was evaluated. Pre-incubation of Cp. psittaci with 0.5 to 5 mg/mL ovoTF prior to infecting BGM cells significantly lowered the infection rate (P < 0.05). For both lactoferrins, the infection rate could only be reduced with 5 mg/mL, albeit not significantly as compared to the infection rate created by the untreated bacteria. Secondly, transferrins were tested for their ability to influence bacterial adhesion and entry in HD11 cells. Maximal non-cytotoxic and non-bactericidal concentrations of 0.05 mg/mL ovoTF and 0.5 mg/mL hLF and bLF were used. Overall, ovoTF was more effective than human and bovine LF in inhibiting bacterial irreversible attachment and cell entry and the latter was accompanied by a dose-dependent reduction of actin recruitment at the bacterial entry site. However, once bacteria had entered HD11 cells, transferrins had apparently no effect on intracellular replication. The present findings suggest a possible role for transferrins and especially ovoTF, in preventing avian Cp. psittaci infections.     

不同浓度的IGF-1对山羊毛囊干细胞体外培养的影响

采用2.4 U/mL Dispase酶消化和机械切割法分离毛囊隆突部,经胰酶（0.5 mg/mL胰酶+0.2 mg/mL EDTA）消化,从山羊耳部皮肤分离得到毛囊干细胞,并检测了不同浓度的IGF-1对山羊毛囊干细胞体外培养的作用。结果表明,IGF-1对毛囊干细胞体外培养作用很明显,并且最适IGF-1浓度是10 ng/mL。     

3种中药及其提取物体外抗猪繁殖与呼吸综合征病毒作用的研究

为研究板蓝根、黄芪和青蒿及其提取物体外抗猪繁殖与呼吸综合征病毒（PRRSV）的作用,本试验在研究板蓝根、黄芪和青蒿及其提取物对Marc-145细胞最大安全浓度的基础上,研究3种中药提取物体外抗PRRSV的作用。结果显示,板蓝根多糖、黄芪多糖和青蒿素3种中药提取物对Marc-145细胞安全浓度分别为为0.03、0.03和0.06 mg/mL,板蓝根、黄芪和青蒿3种中药单方的安全浓度分别为6.25、6.25和3.13 mg/mL。在体外抗PRRSV作用的试验中,3种中药单方中黄芪的阻断作用最强,能达到100%的保护率,中药提取物中板蓝根多糖对PRRSV的阻断作用最强,在浓度为0.031 mg/mL时对细胞保护率能达到48.03%,青蒿素最差,在0.16 mg/mL时就已不具有阻断作用。板蓝根多糖对病毒不具有直接杀灭作用,黄芪多糖和青蒿素在浓度为0.32 mg/mL时,保护率都是20%左右;黄芪的直接杀灭作用保护率为100%,且非常稳定,青蒿在浓度较高时还有促进细胞生长的作用,但稳定性差,随浓度降低其保护率迅速下降。在对病毒的抑制作用试验中,3种中药提取物的作用相当,浓度为0.01 mg/mL时,板蓝根多糖、黄芪多糖和青蒿素多糖对细胞的保护率分别为24.57%、24.30%和26.20%;而3种中药单方的细胞保护率都能达到100%甚至以上。综合3种作用方式可知,3种中药提取物中,黄芪多糖的抗PRRSV作用最好,青蒿素次之,板蓝根多糖最差;3种中药单方中黄芪的效果最好,青蒿最差。因此,可知黄芪及其提取物具有非常强的抗PRRSV作用,可对其进行进一步研究,以供临床上使用。     

Antiviral Effects of Three Chinese Herbal Medicine and Their Polysaccharides on Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in vitro

In order to figure out the antiviral effects of Radix Isatidis,Astragalus herb,Artemisia annua and their polysaccharides on porcine reproductive and respiratory syndrome virus (PRRSV) in vitro,the antiviral activity of the three Chinese herbal medicine and their polysaccharides,Isatis root polysaccharide (IRPS),Astragalus polysaccharides (APS) and artemisinin maximum were evaluated by adding to Marc-145 cell cultured with PRRSV respectively.The results showed that the safety concentrations of IRPS,APS and artemisinin to Marc-145 cells were 0.03,0.03 and 0.06 mg/mL;The safety concentrations of Radix Isatidis,Astragalus herb,Artemisia annua to Marc-145 cells were 6.25,6.25 and 3.13 mg/mL.Almost all the cells (about 100%) were protected from PRRSV when Astragalus herb were added early to PRRSV,while IRPS had the same effect on PRRSV among three extracts with 48.03% cells were protect and artemisinin could not protect cells when the concentration was lower than 0.16 mg/mL.IRPS had no effect on PRRSV while APS and artemisinin got a protection rate for cells at about 20% when simultaneously added with PRRSV,under the concentration of 0.32 mg/mL.In terms of simultaneously added with PRRSV,Chinese herb Astragalus could exterminate the virus directly with its protect cells coming to 100% and more.Artemisia annua could promote the growth of cells but its stability was weak.Its protection rate decreased as its concentration reducing.In the experiment of exploring the antivirus effect of infected cells,polysaccharides of these three herbs had nearly the same effect.When in a concentration of 0.01 mg/mL,IRPS,APS and artemisinin respectively protected 24.57%,24.30% and 26.20% cells.However,the protection rate to cells of the three herbs themselves was respectively up to 100% or more.In conclusion,in the extracts of the three herbs,APS had the best antiviral effect,followed by artemisinin and IRPS,and in the single herbal medicines,Astragalus was the best and Artemisia annua was worst.Therefore,Astragalus and APS had the best antiviral effect on PRRSV.It needed more research on Astragalus and APS,and try to use them as antiviral drug in clinical application.     

山羊毛囊干细胞分离、培养及鉴定

采用2．4U／mLDispase酶消化和机械切割法分离毛囊隆突部,经胰酶（0．5mg／mL胰酶＋0．2mg／mLEDTA）消化从山羊耳部皮肤分离得到毛囊干细胞,用自制无血清培养基培养,并用形态学观察、细胞生长曲线、克隆形成率及免疫组化染色检测,探讨毛囊干细胞体外分离培养方法及生物学特性。结果表明,所分离细胞具备毛囊干细胞特征：体积较小、表面光滑、立体感强,呈现未分化细胞特征;K19、integrin-β1以及p63免疫组化染色阳性;第3、5、7代干细胞克隆形成率分别为25．6％&#177;2．6％、31．8％&#177;1．9％和27．0％&#177;3．9％,极显著高于对照组角质细胞克隆形成率（P〈0．01）。采用配制的无血清条件培养液可使山羊毛囊干细胞体外维持未分化状态并传代至19代。     

Evaluation of the precision of intradermal injection of control substances for intradermal testing in clinically normal horses

OBJECTIVE: To evaluate the precision of intradermal testing (IDT) in horses. ANIMALS: 12 healthy adult horses. PROCEDURE: IDT was performed on the neck of each horse by use of 2 positive control substances (histamine and phytohemagglutinin [PHA]) and a negative control substance. An equal volume (0.1 mL) for each injection was prepared to yield a total of 20 syringes ([4 concentrations of each positive control substance plus 1 negative control substance] times 2 positive control substances times 2 duplicative tests) for each side of the neck. Both sides of the neck were used for IDT; therefore, 40 syringes were prepared for each horse. Hair was clipped on both sides of the neck, and ID injections were performed. Diameter of the skin wheals was recorded 0.5, 4, and 24 hours after ID injection. RESULTS: Intra- and interhorse skin reactions to ID injection of histamine and PHA resulted in wheals of uniform size at 0.5 and 4 hours, respectively. Significant intra- and interhorse variation was detected in wheals caused by PHA at 24 hours. CONCLUSIONS AND CLINICAL RELEVANCE: ID injection of histamine and PHA caused repeatable and precise results at 0.5 and 4 hours, respectively. Concentrations of 0.005 mg of histamine/mL and 0.1 mg of PHA/mL are recommended for use as positive control substances for IDT in horses. This information suggests that consistent wheal size is evident for ID injection of control substances, and variation in wheals in response to ID injection of test antigens results from a horse's immune response to specific antigens.     

Plasma pharmacokinetics, pulmonary distribution, and in vitro activity of gamithromycin in foals

The objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Rhodococcus equi. A single dose of gamithromycin (6 mg/kg of body weight) was administered intramuscularly. Concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils were determined using HPLC with tandem mass spectrometry detection. The minimum inhibitory concentration of gamithromycin required for growth inhibition of 90% of R. equi and S. zooepidemicus isolates (MIC(90)) was determined. Additionally, the activity of gamithromycin against intracellular R. equi was measured. Mean peak gamithromycin concentrations were significantly higher in blood neutrophils (8.35±1.77 μg/mL) and BAL cells (8.91±1.65 μg/mL) compared with PELF (2.15±2.78 μg/mL) and plasma (0.33±0.12 μg/mL). Mean terminal half-lives in neutrophils (78.6 h), BAL cells (70.3 h), and PELF (63.6 h) were significantly longer than those in plasma (39.1 h). The MIC(90) for S. zooepidemicus isolates was 0.125 μg/mL. The MIC of gamithromycin for macrolide-resistant R. equi isolates (MIC(90)=128 μg/mL) was significantly higher than that for macrolide-susceptible isolates (1.0 μg/mL). The activity of gamithromycin against intracellular R. equi was similar to that of azithromycin and erythromycin. Intramuscular administration of gamithromycin at a dosage of 6 mg/kg would maintain PELF concentrations above the MIC(90) for S. zooepidemicus and phagocytic cell concentrations above the MIC(90) for R. equi for approximately 7 days.