HPLC purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis

The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatography. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein-immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The relative sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.     

Isolation and identification of F-spondin in the boar testis and its production during testis growth

F-spondin/vascular smooth muscle cell growth-promoting factor (VSGP), purified from the follicular fluid of adult bovine ovaries, has been identified as a promoter of neuronal differentiation and vascular smooth muscle growth. The objectives of the present study were (1) to clarify whether F-spondin is also produced in the testis, which is ontogenically equivalent to the ovary, and (2) to examine whether production of this protein changes with testicular growth. To isolate F-spondin from the testis, testicular homogenates obtained from 8-week-old boars were sequentially subjected to heparin-Sepharose chromatography, diethylaminoethyl (DEAE)-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The isolated protein had a molecular mass of approximately 110 kDa and was cross-reactive with anti-F-spondin antibody by Western blotting. The purified protein was further characterized by amino acid sequence analysis of its internal peptide. The sequence obtained was GEQCNIVPDN VD, and a homology search indicated that the purified protein is a homologue of rat, human, and bovine F-spondin. By fractionation of the same amounts of testis tissue obtained from 1-, 8-, 16-, and 40-week-old boars, we analyzed age-related production of F-spondin in the testis. Western blotting of the fractions obtained from RP-HPLC revealed the presence of a band at approximately 110 kDa, corresponding to F-spondin, in the testes obtained from boars between 1 and 16 weeks old, but this band was not detected at 40 weeks. These results clearly indicate that (1) the porcine testis produces F-spondin and that (2) production of this protein is evident in the immature porcine testis, but not the adult testis.     

Detection of anticoagulant rodenticides (4-hydroxycoumarins) by thin-layer chromatography and reversed-phase high-performance liquid chromatography with fluorescence detection

The detection of 4-hydroxycoumarin rodenticides in poisoned domestic animals requires a highly sensitive method as tissue and serum levels of anticoagulants may be very low owing to rapid elimination, metabolism or post-mortem degradation. Thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence detection were used to identify the anticoagulants in spiked tissues and in suspicious samples. The analysis of ten suspicious samples highlighted the limitations of both methods. Only the three samples of baits were found positive by TLC whereas one of the five anticoagulants was detected in eight samples by RP-HPLC with fluorescence detection. Therefore, RP-HPLC with fluorescence detection proved to be the more sensitive method for detecting low levels of 4-hydroxycoumarins in blood serum, liver and ingesta, whereas TLC is usually sufficient for analysing baits.Abbreviations RP-HPLC reversed-phase high-performance liquid chromatography - TLC thin-layer chromatography     

乳清蛋白抗菌肽的分离纯化及活性

以乳清蛋白为原料,酶解制备具有抗菌能力的生物活性肽。采用超滤、葡聚糖凝胶层析色谱、反相高效液相色谱法对酶解液进行分离纯化,并对分离产物的氨基酸组成进行分析。结果表明:葡聚糖凝胶色谱纯化最佳流速2 mL/min,最佳样品质量浓度15 mg/mL;再由反相高效液相色谱分离的高活性抗菌肽,抑菌圈直径达到31.33 mm。氨基酸分析结果显示,高活性抗菌肽碱性氨基酸和疏水性氨基酸含量最多,分别为42.69%和37.39%。     

猪链球菌2型江苏分离株溶血素的纯化

将猪链球菌2型(Streptococcus suis type2)江苏分离株HA9801接种于THB培养基中培养，得到含溶血素的培养液，其溶血价为128，再经50％饱和硫酸铵沉淀，脱盐，得到溶血素粗提物，溶血价为2048。粗提物用阴离子交换柱层析及凝胶过滤层析，所得活性峰收集液溶血价分别为4096、1024。通过以上三步的纯化，溶血素的比活提高200倍。纯化蛋白在SDS-PAGE中呈现一条带，达电泳级纯度。     

Use of enzyme immunoassay and reverse-phase high-performance liquid chromatography to detect and confirm identity of dexamethasone in equine blood.

An enzyme immunoassay (EIA) was developed for detection of dexamethasone in equine blood. Dexamethasone 21-hemisuccinate-bovine serum albumin was used for immunization of rabbits, and prednisolone 21-hemisuccinate-horseradish peroxidase was used as enzyme conjugate. The assay had sensitivity in the low-picogram range (detection limit, 0.3 pg/well, 50% inhibition of binding at 4.5 +/- 0.7 pg/well). Apart from cortisol, which was recognized by the antiserum at concentration > 8.5 ng/ml, the dexamethasone antiserum failed to interfere with endogenous steroids, but cross-reacted with triamcinolone, flumethasone, and betamethasone. Thus, the antiserum was used to perform simultaneous screening for these synthetic glucocorticoids and to confirm their identity by combining reverse-phase high-performance liquid chromatography (RP-HPLC) and EIA. The immunoreactivity obtained by direct serum measurements was characterized by means of 2 independent RP-HPLC systems. Serum extracts were submitted to RP-HPLC systems I and II, and the fractions were tested by EIA. Immunoreactive peaks were identified by comparing their retention time with that of the standard glucocorticoids used for calibration. Coinjection of an internal standard (methylprednisolone) in RP-HPLC system II yielded reproducible relative retention times. The effectiveness of the test system was evaluated, using blood from a horse treated with commonly used veterinary preparations of dexamethasone. Administration of the free alcohol of dexamethasone and of dexamethasone 21-trioxaundecanoate, both given IV, was detected, and the identity of each was confirmed for up to 48 hours. Intramuscular administration of dexamethasone 21-isonicotinate was continued for at least 14 days after injection of a therapeutic dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of bovine ovarian inhibin, its immunoneutralization in vitro and immunolocalization in bovine ovary

A purification scheme involving gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) was used to isolate from bovine follicular fluid (FF) biologically-active inhibin of molecular weight 32 kDa. Chromatographic fractions were monitored for inhibin-like biological activity (ILA) using a simplified bioassay procedure in which a suppression of total basal FSH production by rat pituitary cells in monolayer culture indicates the presence of ILA. Approximately 3 mg protein having an ILA potency (ED50 value in in vitro bioassay) of 1.7 ng/ml was obtained from 4 1 crude bovine FF (260 g protein; ILA potency 3750 ng/ml) reflecting an approximate 2200-fold purification factor with an overall recovery of about 3%. The isolated material appeared as a single major UV absorbance peak on RP-HPLC and as a single band (32 kDa) when subjected to SDS-PAGE (15% gel) under non-reducing conditions. Under reducing conditions the molecule dissociated into 2 subunits of apparent molecular weight 22 and 14 kDa confirming that it is probably identical to the 31/32 kDa form of bovine ovarian inhibin previously reported by two other independent research groups. An antiserum raised in a chicken against the isolated material completely neutralized the suppressive effects of both 32 kDa inhibin and bovine FF on basal production of FSH by rat pituitary cells in vitro but only partially reversed the suppressive effects of both porcine and human FF. Immunohistochemical staining of sections of bovine ovary and of isolated preparations of bovine granulosa cells using this antiserum confirmed that granulosa cells are a major source of inhibin. The observation that specific immunostaining was not confined to these cells, however, suggests that they may not be the exclusive source of immunoreactive inhibin in the bovine ovary.     

Purification and initial characterisation of koala immunoglobulins

The production of koala immunoglobulins (Ig) was elicited by the immunisation of a koala (Phascolarctos cinereus) with bovine serum albumin. This Ig was then purified using the highly specific techniques of affinity chromatography. The purified protein was compared with a "potential" koala Ig protein which was subsequently purified by Protein G chromatography and both proteins were further characterised by agarose/SDS-PAGE and immunoelectrophoresis. Results indicate that koalas do produce Ig in response to antigen challenge, koala Ig has a higher net negative charge than that seen in most other mammals and possibly two subclasses of IgG and IgM are present in normal koala serum.     

三聚氰胺在猪体内的药物残留研究

此研究通过采用养殖场饲料配方喂养猪,在不同时间通过反相高效液相色谱法测定三聚氰胺在猪的背肌、腿肌、肝脏、肺、肾脏、心脏、血尿中的残留量,用以研究三聚氰胺在猪体内的残留特征和代谢规律。结果表目月．三聚氰胺在猪体内为惰性代谢,大部分以原型从尿液排出,大剂量应用时主要对肾脏产生毒性,同时对心脏等肾脏以外的器官也产生毒性作用。三聚氰胺在猪体内消除缓慢,残留期较长,基本残存时间在15d左右。     

三种方法纯化相思子毒素单抗的比较

采用不同的方法纯化相思子毒素单抗,寻求一种效果好、操作简便的纯化方法。采用硫酸铵沉淀法、辛酸-硫酸铵法和硫酸铵-蛋白A亲和层析法三种方法分离纯化相思子毒素单抗,并采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、BCA蛋白检测试剂盒(BCA Protein Assay Kit)和双抗夹心酶联免疫吸附试验(ELISA)对纯化产物进行相对分子质量、浓度、纯度、回收率及免疫活性的鉴定。结果表明,纯化后单抗的回收率以硫酸铵沉淀法及辛酸-硫酸铵法较高,硫酸铵-蛋白A亲和层析法较低;产物纯度以硫酸铵-蛋白A亲和层析法和辛酸-硫酸铵法较高,硫酸铵沉淀法较差;不同方法纯化后的单抗活性未见降低。     

牛β-酪蛋白多克隆抗体的制备及鉴定

以牛β-酪蛋白标准品为免疫原,免疫健康的新西兰大白兔,制备了兔抗牛β-酪蛋白的多克隆抗体。经亲和层析纯化后,用SDS-PAGE电泳及考马斯亮蓝染色检测抗体的纯度,经ELISA法测定β-酪蛋白抗体的效价为1∶1600。Western-blot分析发现,制备的抗体与β-酪蛋白可以发生特异性结合,表明成功制备了纯度较高和特异性较好的牛β-酪蛋白多克隆抗体,为乳腺生物反应器的研究提供了有效的工具。     

低分子质量左聚糖对人体肠道菌群调节作用

采用三氯乙酸(trichloroacetic acid,TCA)法除去Paenibacillus bovis sp.nov BD3526来源的高分子质量左聚糖中蛋白质的同时制备得到低分子质量左聚糖,并用离子交换层析法对低分子质量左聚糖进行分离纯化,用凝胶渗透色谱(gel permeation chromatography,GPC)法测定纯化后样品的分子质量及其分布,用核磁共振(nuclear magnetic resonance,NMR)仪和傅里叶变换红外光谱(fourier transform infrared spectrometer,FT-IR)表征其化学结构,用原子力显微镜(atomic force microscope,AFM)表征其在水溶液中的构象,最后采用荷兰应用科学研究(The Netherlands Organization For Applied Scientific Research,TNO)组织建立的体外筛选平台评价其对人体肠道菌群的调节作用.体外实验结果表明,此低分子质量左聚糖能促进双歧杆菌特别是婴儿双歧杆菌和长双歧杆菌的增殖,其促进作用优于低聚果糖(fructooligosaccharide,FOS).     

兔出血症病毒提纯和多肽的比较研究

我们采用了甘油－酒石酸钿梯度离心、琼脂糖４Ｂ层析和ＤＥＡＥ３２离子交换层析等三种方法提纯兔出血症病毒（ＲＨＤＶ）进行比较，其结果发现ＤＥＡＥ３２离子交换层析效果最好，获得的病毒纯度最高，经ＳＤＳ－ＰＡＧＥ和Ｗ－Ｂｌｏｔ试验表明，ＲＨＤＶ只有一条蛋白多肽分子量为６１ＫＤ，截然不同于以往国内研究报道的ＲＨＤＶ具有３－６条结构多肽等结果，在国内属首次报道。     

牦牛肉中游离氨基酸含量的分析

采用反相高效液相色谱法测定了7头麦洼牦牛后腿肌肉中游离氨基酸的含量。在牦牛肉中检测到16种游离的标准氨基酸,总含量为532.34±86.74 mg/100 g,其中含量最高的游离氨基酸为精氨酸,其次为组氨酸,这两种氨基酸约占游离氨基酸总量的75%。     

Development of a feline proinsulin immunoradiometric assay and a feline proinsulin enzyme-linked immunosorbent assay (ELISA): a novel application to examine beta cell function in cats

The prevalence of feline diabetes mellitus has increased several-fold over the last three decades. In humans, progression from obesity to diabetes is marked by changes in the release of proinsulin. A specific proinsulin (FPI) assay has not been available to examine similar changes in cats. The goal of this study was to develop a proinsulin assay for the analysis of beta cell function in cats. Monoclonal antibodies were developed against recombinant FPI and used in a two-site sandwich immunoradiometric assay (IRMA) and enzyme-linked immunosorbent Assay (ELISA). The antibody pair had negligible cross-reactivity with bovine insulin and feline C-peptide. The working range was 11-667pmol/L for the IRMA and 11-1111pmol/L for the ELISA. An intravenous glucose tolerance test was performed in six long-term obese and six lean adult healthy cats and serum glucose, insulin, and FPI concentrations were determined. The proinsulin and insulin secretion pattern in response to glucose was significantly different between lean and obese cats but the pattern was similar within a group. Both groups had similar baseline proinsulin/insulin ratios; however, obese cats showed a significantly higher proinsulin/insulin ratio during the first 15min of the IVGTT and a much lower ratio during the last 30min suggesting a time-delayed adjustment to the increased insulin demand. In conclusion, we report the development and validation of an IRMA and an ELISA for FPI. This novel assay is useful to elucidate FPI secretion and can be used similar to a C-petide assay to evaluate residual beta cell function in cats.     

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.     

伊氏锥虫谷胱甘酰亚精胺还原酶（TR）的研究（二）

用ＣＥＡＥ ＤＥ－５２分离纯化的锥虫，经超声波粉碎后离心，上清经硫酸铵沉淀，再经Ｓｅｐｈａｄｅｘ Ｇ－２００分子筛层析，ＤＥＡＥ Ｃｅｌｌｕｌｏｓｅ ＥＤ－５２离子交换层析，２’，‘ＡＤＰ－Ｓｅｐｈａｒｏｓｅ层析得到纯化。筛选到抗蠕虫药奥芬哒唑（Ｏｘｆｅｎｄａｚｏｌｅ）对ＴＲ有抑制作用。但对治疗锥虫病没有作用；兔抗ＴＲ抗体与安锥赛共同注射人工感染锥虫病的小白鼠，可增加安锥赛对锥虫病的疗效。     

Autoantibodies to recombinant canine proinsulin in canine diabetic patients

Objective

To determine whether dogs with spontaneously-occurring diabetes mellitus demonstrate serological reactivity to proinsulin.

Sample population

Serum samples were collected from 15 newly-diagnosed diabetic, 15 insulin-treated diabetic and 15 non-diabetic control dogs.

Procedures

Canine proinsulin was cloned into a prokaryotic expression vector to generate recombinant poly-histidine-tagged protein in Escherichia coli. A Western blotting assay was developed for detection of proinsulin autoantibodies in canine sera.

Results

Reactivity to canine proinsulin was detected in 3 of 15 control dogs, 8 of 15 newly-diagnosed diabetic dogs and 6 of 15 insulin-treated diabetic patients. Of these reactors, only 1 control dog, 1 newly-diagnosed diabetic dog and 3 insulin-treated diabetic dogs recognised porcine insulin by ELISA, suggesting that the remaining proinsulin reactors might have been recognising proinsulin-specific epitopes.

Conclusions and clinical relevance

This study suggests that proinsulin autoantibodies are present in a proportion of diabetic dogs. Further work is required to refine the assay and clarify the significance of these autoantibodies.     

用反相高效液相色谱-紫外检测法测定桑叶中芦丁、槲皮素和1-脱氧野尻霉素的含量

为高效检测桑叶中的药用活性成分,进行了用反相高效液相色谱-紫外检测法(RP-HPLC-UV)综合测定桑叶中芦丁、槲皮素和1-脱氧野尻霉素(1-DNJ)含量的试验。采用85%乙醇超声辅助萃取桑叶中的芦丁和槲皮素,以0.05 mol/L HCl为溶剂提取1-DNJ,并在pH8.5的硼酸盐缓冲液中进行衍生化反应,生成具有紫外吸收的1-DNJ-FMOC络合物。色谱柱为Alltima C18(4.6 mm×250 mm,5μm),检测波长分别为360、360、254 nm,流动相分别为V(四氢呋喃)∶V(0.02%磷酸)=25∶75、V(乙腈)∶V(pH4.0、0.5 mol/L醋酸缓冲液)=27∶73、V(乙腈)∶V(0.1%醋酸)=36∶64。检测结果显示:芦丁、槲皮素和1-DNJ的色谱图分离效果良好,峰面积与浓度呈高度正相关,回收率(n=3)分别为99.14%、98.89%、100.85%,相对标准偏差(RSD)分别为1.01%、0.33%、1.24%。试验显示建立在以上条件下的检测方法精密度高,检测结果准确可靠。     

鸡免疫球蛋白G Fc(IgG Fc)片段的分离纯化及其抗血清的制备 Ⅱ.豚鼠抗鸡IgG Fc片段血清的制备

经硫酸铵盐析、DEAE 32-纤维素和Sephadex G 200柱色谱法分离得到纯化的鸡血清IgG。然后用木瓜蛋白酶水解IgG,再经DEAE 32-纤维素柱色谱纯化制得IgG Fc片段,并以IgG Fc片段免疫豚鼠制备豚鼠抗鸡IgG Fc血清。