猪表皮生长因子间接竞争ELISA检测方法的建立

为建立酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测猪血清或尿液中猪表皮生长因子(porcinee pidermal growth factor,pEGF)的含量,以pEGF为包被原,人表皮生长因子(hu-mane pidermal growth factor,hEGF)为竞争抗原,两者与一定量的抗pEGF多抗反应。结果表明,理想的包被抗原浓度为0.625μg/mL,抗pEGF工作浓度为1∶64000,酶标二抗工作浓度为1∶20000,可检测的最适范围为0.4ng/mL~32ng/mL,最低检测限为1ng/mL,批内和批间变异系数分别为3.50%和5.22%。得到回归方程y=-34.53x 76.06(R2=0.993)和标准曲线,从而建立了快速定量测定pEGF含量的酶联免疫吸附试验方法。     

大豆抗原蛋白血清抗体间接ELISA检测方法的建立

旨在建立检测血清大豆抗原蛋白抗体的间接ELISA方法。经琼脂糖凝胶层析纯化大豆抗原蛋白,以不同剂量皮下注射免疫小鼠,采用方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其他条件进行优化,最终建立检测血清大豆抗原蛋白抗体的间接ELISA方法,利用该方法检测小鼠免疫后血清抗体水平。通过方阵滴定法确定11S蛋白最佳包被浓度为5.0μg/mL,血清稀释倍数为1∶800;7S蛋白抗原最佳包被浓度为2.5μg/mL,血清稀释倍数为1∶1 600;两者的批内、批间系数均小于10%,重复性较好,通过ELISA法确定11S和7S蛋白的最佳免疫次数为2次,免疫剂量为1 000μg/kg。结果表明本试验初步建立大豆抗原蛋白抗体检测间接ELISA方法,具有很强的特异性、敏感性和重复性,可用于大豆抗原蛋白过敏反应的临床检测。     

青霉素酶特异性抗血清间接ELISA方法的建立

以青霉素酶为抗原,免疫健康獭兔,自行制备青霉素酶特异性抗血清(PcAb),并建立间接酶联免疫吸附试验(ELISA)的测定方法,优化试验条件,测定制备的青霉素酶特异性抗血清效价。结果表面抗原最佳包被浓度为10μg/mL,酶标二抗最佳稀释度为1∶2 000,阳性血清最佳稀释度为1∶2 000,抗原最佳包被条件为4℃过夜,血清最佳反应时间为37℃1 h,酶标二抗反应最佳时间为37℃1 h,与底物作用的最佳时间为20 min。     

牛奶中替米考星间接竞争酶联免疫吸附检测法的建立

本研究通过棋盘法优化替米考星抗体、包被原浓度,并考察了最佳包被条件,最佳反应温度、时间和酶标二抗最佳反应时间等,建立了替米考星残留的间接竞争酶联免疫吸附试验(ELISA)并应用到牛奶样本的检测。建立的间接竞争ELISA方法半数抑制浓度(IC₅₀)为2.1 ng/mL,牛奶中替米考星的最低检测限(LOD)为2 μg/L。在5、10和20 μg/L添加浓度下,回收率为79.2%~90.1%,变异系数不高于9.0%。与其他常见的大环内酯类药物无交叉反应。本研究建立的替米考星间接竞争ELISA方法灵敏度达到了残留限量标准要求,为开发可用于牛奶中替米考星的快速检测试剂盒奠定基础。     

间接ELISA检测抗氨苄青霉素抗体方法的建立

通过对最佳抗原包被浓度及包被条件、最佳血清工作浓度、封闭剂、待测血清、酶标二抗的最佳工作浓度及作用时间等反应体系的筛选和确定,利用人工制备的完全抗原及其免疫血清建立了检测抗氨苄青霉素(AMPI)抗体的间接ELISA 方法。结果表明,AMPI BSA抗原最佳包被浓度为1μg/mL,AMPI HSA抗原最佳包被浓度为2μg/mL;包被条件为37 ℃4 h 后4 ℃过夜;抗AMPI HSA血清和抗AMPI BSA 血清最佳工作浓度分别为1∶25 600 和1∶51 200;封闭剂、待测血清、酶标二抗的最佳作用时间均为30 min。血清检测结果表明抗AMPI HSA的抗体水平最高,抗AMPI BSA 的抗体水平稍低于抗AMPI HSA 的抗体,而抗AMPI KLH的最低。     

食品中呋哺妥因代谢物酶联免疫检测试剂盒的研制

目的:开发研制快速检测呋喃妥因代谢物的酶联免疫检测试剂盒.方法:依据直接竞争ELISA原理,采用棋盘包被法确定最佳抗体包被浓度和酶标抗原工作浓度、包被条件、反应时间、底物显色时间等,并对试剂盒的各项技术指标进行确认.结果:成功的组装了呋喃妥因的酶联免疫检测试剂盒,并建立了肌肉、肝脏、水产、蜂蜜等的前处理方法,检测限均远低于1μg／kg.该试剂盒线性检测范围为0～4.05ng／mL,IC50浮动范围0.30～0.60ng/mL,样品的板内、批内、批间的变异系数均小于15％,平均回收率在65％～90％之间,与其同类药物的交叉反应率均小于0.2％.结论:本研究研制的试剂盒重复性、再现性、特异性、稳定性各项指标均符合技术要求,可用于动物源性食品中呋喃妥因代谢物残留的检测.     

山羊IFN-γ免疫血清制备及抗体ELISA检测方法的建立

为制备山羊干扰素-γ(IFN-γ)免疫血清并建立山羊IFN-γ抗体ELISA检测方法 ,以原核表达的山羊γIFN重组蛋白(rIFN-γ)为材料,免疫小鼠制备免疫血清;通过对测定不同的抗原包被浓度及包被时间,酶标抗体稀释度,免疫血清的孵育时间等条件的优化建立检测抗体效价的ELISA检测方法,并采用建立的方法测定免疫血清的抗体效价。结果显示,rIFN-γ抗原包被浓度为10μg/mL,4℃包被12h;酶标抗体稀释度为1∶5 000;免疫血清孵育时间为60min,可以得到最佳ELISA的检测结果。重复试验显示该方法变异系数(CV)在1.5%~5%之间。采用建立的方法测得免疫小鼠免疫血清效价为107。该ELISA检测方法灵敏高,稳定性好,为山羊IFN-γ抗体检测提供了技术支持。     

猪繁殖与呼吸综合征病毒Nsp7α抗体ELISA检测方法的建立

本研究克隆、表达了猪繁殖与呼吸综合征病毒DY株(GenBank:JN864948)的Nsp7α蛋白,并利用Nsp7α作为包被蛋白建立了ELISA检测方法,并对检测条件进行了优化。结果显示,ELISA最佳条件:抗原包被浓度为1μg/mL,血清稀释度为1∶40,封闭液为10%牛血清,酶标二抗工作稀释度为1∶4000,显色时间为37℃反应15 min。用建立的ELISA检测方法和IDEXX ELISA检测试剂盒检测血清样品115份,总符合率为93.91%。本研究中Nsp7α抗体ELISA检测方法的建立,为PRRSV的抗体监测提供了新的技术支持。     

恩诺沙星ELISA快速检测方法的建立

采用活酯法合成酶标记半抗原（ENR-HRP），紫外扫描分析和ELISA方法鉴定，结合比为1．6：1。选择ENR-HRP与游离ENR相竞争的模式建立检测ENR残留的ELISA检测方法，其最佳工作条件为：二抗的包被浓度为2μg／mL，抗体工作浓度1：1000，酶标半抗原工作浓度1：500。标准曲线在0．423～1000ng／mL之间线性关系良好，其IC50为33．76ng／mL，回归方程为Y=-0．07769lnx＋0．770801，相关系数r=0．99153，最低检测限0．423ng／mL。单抗对达氟沙星、培氟沙星、麻保沙星有少许交叉，特异性较好。ELISA检测方法批内平均变异系数为5．75％，批间平均变异系数为10．69％，重复性较好。     

达氟沙星间接竞争ELISA检测方法的建立

将达氟沙星（danofloxacin, DFLX）与牛血清白蛋白(BSA)、卵清白蛋白(OVA)偶联分别作为免疫原与包被原, 达氟沙星为竞争的半抗原，建立间接竞争ELISA检测方法。试验结果表明,理想的包被抗原浓度为1.25 μg/L, 多抗(DFLX-PcAb)工作浓度为1∶10000,酶标二抗的工作浓度为1∶4000,最适检测范围为0.1～10 ng/L,最低检测限为0.2 ng/L, 批内和批间变异系数分别为3.81%和6.25%。得到回归方程y=0.7975-0.125x（R2=0.989）和标准曲线，从而建立了DFLX的快速检测残留的间接竞争酶联免疫吸附试验（ci-ELISA）。     

商会自治的基石——商会自治规范研究

在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。     

癸氧喹酯干混悬剂含量测定的改进

采用高效液相色谱法测定癸氧喹酯干混悬剂的含量,在2-250μg/mL范围内,峰面积的常用对数与进样量浓度的常用对数呈良好的线性关系,R^2=1(n=5),平均回收率为99.24%~99.51%,RSD在0.05%~0.28%。此方法分析时间短,样品前处理简便、定量结果准确,重现性好,结果满意,为其质量控制提供了依据。     

猪毛色遗传的研究进展

本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。     

Comparison of 2 methods of centesis of the bursa of the biceps brachii tendon of horses

REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air.     

不同样本商品蜂蜜中硒含量的测定

用硝酸和高氯酸消化蜂蜜,使硒游离出来,在微酸性环境下,硒和2,3-二氨基萘(DAN)生成有较强荧光的物质,用环己烷萃取,在激发波长378nm,荧光波长518nm处测定其荧光强度。蜂蜜中硒含量范围:0.10～0.82μg/g。表明:蜂蜜应视为天然富硒营养品。     

乳酸杆菌益生作用机制的研究进展

乳酸杆菌作为益生菌广泛用于人和动物。本文综述了乳酸杆菌改善宿主健康的机制。乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道。文章重点讨论了乳酸杆菌表面成分（表面蛋白、脂磷壁酸和肽聚糖）与肠道受体（Ｃ型凝集素受体、Ｔｏｌｌ样受体和 Ｎｏｄ样受体）,阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制。     

Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo.     

Mechanism of Action of Probiotic Function of Lactobacilli

乳酸杆菌作为益生菌广泛用于人和动物.本文综述了乳酸杆菌改善宿主健康的机制.乳酸杆菌可通过产生抗菌物质如乳酸、过氧化氢、细菌素,或者通过竞争营养或肠道黏附位点来抑制致病菌;通过诱导黏附素的分泌或阻止细胞凋亡而增强肠道的屏障功能,从而保护肠道.文章重点讨论了乳酸杆菌表面成分(表面蛋白、脂磷壁酸和肽聚糖)与肠道受体(C型凝集素受体、Toll样受体和Nod样受体),阐述了他们结合后启动免疫调节信号,调控肠道免疫功能以发挥改善健康作用的机制.     

Evaluation of serum concentrations of biomarkers of skeletal metabolism and results of radiography as indicators of severity of osteochondrosis in foals

OBJECTIVE: To determine whether serum concentrations of biomarkers of skeletal metabolism can, in conjunction with radiographic evaluation, indicate severity of osteochondrosis in developing horses. ANIMALS: 43 Dutch Warmblood foals with varying severity of osteochondrosis. PROCEDURE: 24 foals were monitored for 5 months and 19 foals were monitored for 11 months. Monthly radiographs of femoropatellar-femorotibial and tibio-tarsal joints were graded for osteochondral abnormalities. Serial blood samples were assayed for 8 cartilage and bone biomarkers. At the end of the monitoring period, foals were examined for macroscopic osteochondrosis lesions. RESULTS: Temporal relationships were evident between certain serum biomarkers and osteochondrosis severity in foals during their first year. Biomarkers of collagen degradation (collagenase-generated neoepitopes of type-II collagen fragments, type-I and -II collagen fragments [COL2-3/4C(short)], and cross-linked telopeptide fragments of type-I collagen) and bone mineralization (osteocalcin) were positive indicators of osteochondrosis severity at 5 months of age. In foals with lesions at 11 months of age, osteochondrosis severity correlated negatively with COL2-3/4C(short) and osteocalcin and positively with C-propeptide of type-II procollagen (CPII), a collagen synthesis marker. Radiographic grading of osteochondrosis lesions significantly correlated with macroscopic osteochondrosis severity score at both ages and was strongest when combined with osteocalcin at 5 months and CPII at 11 months. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of serum biomarkers to indicate osteochondrosis severity appears to depend on stage of disease and is strengthened with radiography. In older foals with more permanent lesions, osteochondrosis severity is significantly related to biomarker concentrations of decreased bone formation and increased cartilage synthesis.