人血清中分离纯化鸡IgG、IgM的实用方法

本试验用绵羊红细胞免疫鸡,免疫后5天采血,制备富集IgM血清。应用硫酸铵粗提、Sepharose-4B分子筛层析方法,从富集IgM的鸡血清一次性分离提取高纯度的IgM及纯度较高的IgG,DEAE－52纤维素离子交换层析进一步纯化,获得电泳纯的IgG。经鉴定该方法制备的IgG,IgM完全可以满足免疫学实验要求,从一份血清样品中同时纯化IgG、IgM两种免疫球蛋白,既省时又节省原料。     

鸭血清IgG的提纯方法比较

本文对鸭血清分别采用了硫酸钠法、辛酸-硫酸铵法、硫酸铵法进行了鸭IgG的提取。提取后的鸭IgG样品用纯度和蛋白含量两个指标来衡量。用辛酸-硫酸铵法提取的鸭IgG样品蛋白含量为9.45mg/ml,纯度能达到用于免疫标记技术的要求,而且操作简单、价格低廉,适于从大批量样品中提纯鸭IgG;用硫酸钠法提取的鸭IgG样品蛋白含量为9.14mg/ml,相对较高,但纯度较低;用硫酸铵法提取的鸭IgG样品蛋白含量、浓度都较低。     

Sensitive detection of PrPSc by Western blot assay based on streptomycin sulphate precipitation

Transmissible spongiform encephalopathies, also termed prion diseases, are fatal neurodegenerative disorders that affect both humans and animals, which are characterized by presences of protease-resistance disease-associated prion protein (PrP(Sc)) in brains. In the present study, we optimized the Western blot assay for PrP(Sc) with a precipitation procedure of streptomycin sulphate. After incubated with suitable amount of streptomycin sulphate, the detective sensitivity for PrP(Sc) was remarkably improved. The precipitation of PrP(Sc) was obviously influenced by pH value in the solution. Employs of PrP(Sc) stock sample into various mimic specimens, including normal hamster brain homogenate, human cerebrospinal fluid and urine, demonstrated that streptomycin precipitation markedly increased the detective sensitivity of PrP(Sc), regardless in low concentration or in large volume. In addition, the PrP(Sc) from a human brain tissue of familiar Creutzfeldt-Jakob disease (fCJD) was efficiently precipitated with streptomycin sulphate. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential, alone or combined with other techniques, to detect low levels of PrP(Sc) in the specimens not only from central nerve system, but also from peripheral organs or fluids.     

HPLC法测定硫酸头孢喹肟冻干粉针中硫酸头孢喹肟含量

建立高效液相色谱法(HPLC法)测定硫酸头孢喹肟冻干粉针中硫酸头孢喹肟的含量。色谱条件为色谱柱Kromasil C18柱(250 mm×4.6 mm,5μm),以高氯酸钠-磷酸-三乙胺缓冲液(pH3.6):乙腈(体积比90∶10)为流动相,流速为0.8 mL/min,紫外检测波长为254 nm。结果表明,硫酸头孢喹肟在0.05~25μg/mL范围内呈良好的线性关系,r=0.99 96,平均回收率为100.34%(n=9),RSD为1.34%。该方法简便快速,结果准确可靠,可用于硫酸头孢喹肟冻干粉的质量控制研究。     

小鼠腹水IgG类单克隆抗体纯化方法的研究

分别采用辛酸硫酸铵法(CA AS)、硫酸铵沉淀法(AS)和DEAE离子交换层析法对小鼠IgG腹水进行了纯化效果的比较。结果表明:CA AS法提取IgG的纯度为67.5%,高于AS法(43.2%),低于DEAE法(100%);CA AS法提取IgG的回收率为51.2%,远高于DEAE(8.9%),略低于AS法(63.8%)。将不同纯化方法获得的IgG定容至相同浓度。ELISA试验结果表明:DEAE法的OD值明显降低,表明DEAE纯化方法降低了IgG的免疫学活性。说明CA AS法是一种操作简单、成本低、纯化效果和回收效果较佳的提取小鼠腹水IgG的首选方法。     

大白鼠血清IgG的提纯及其抗血清的制备

经硫酸铵及DEAE纤维素柱层析提纯的大白鼠血清IgG免疫家兔制备兔抗鼠IgG高免血清，双向琼指扩散效价为1：64，免疫电泳出现一条特异性沉淀线。其纯度和含量均符合实验要求。     

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。     

冷冻干燥法制备速溶植酸酶的研究

采用毕赤酵母发酵制备酶液,膜超滤浓缩、硫酸铵盐析法沉淀酶蛋白,结合冷冻干燥法制得速溶型植酸酶。结果表明,对酶活力达19 500U/ml的发酵液进行浓缩后,以70%饱和度的硫酸铵进行盐析,分离后的酶蛋白经过冷冻干燥得到活力为500 000U/g的植酸酶,速溶性好,溶解后清亮透明。酶的冻干粉50℃保存90d内酶活力仍能维持96%以上的活力。为高酶活速溶植酸酶的工业化生产提供了一条可行的思路。     

鸡免疫球蛋白(GIgG)轻链的分离纯化及其抗血清的制备

经硫酸铵盐析、柱色谱分离得到纯化的鸡血清IgG。继用2-巯基乙醇还原、碘乙酰胶烷化拆开IgG的轻、重链,再经Sephadex G100凝胶过滤柱色谱分离得到IgG轻链。以IgG轻链免疫家兔制得兔抗鸡IgG轻链抗血清。     

Experimental Edema Disease of Swine (E. Coli ENTEROTOXEMIA) I. Detection and Preparation of an Active Principle

Freeze-thaw lysates prepared from strains of Escherichia coli belonging to serogroups O138, O139, and O141 contained a principle (edema disease principle) which induced edema disease in swine. All freeze-thaw lysates contained endotoxic activity that tended to obscure the edema disease syndrome and methods were developed to reduce such activity. Freeze-thaw lysates prepared from E. coli O139 induced the most characteristic edema disease syndrome. Partially purified edema disease principle prepared from O139 freeze-thaw lysates by sequential precipitation with ammonium sulphate and streptomycin sulphate had increased specific activity with markedly reduced endotoxic activity. This material was insoluble at acidic pH but readily soluble at alkaline pH. The effective molecular weight of edema disease principle, based on retention and filtration properties of diaflo membranes, appeared to be greater than 50,000 and less than 100,000. The biological activity of edema disease principle was thermolabile. Sodium deoxycholate treatment of edema disease principle further reduced endotoxic activity. A thermolabile, ammonium sulphate precipitable material was prepared from E. coli O139 that induced a predictable syndrome which resembled edema disease clinically and pathologically following intravenous inoculation in pigs.     

两种纯化兔抗酸性红73免疫球蛋白G方法的比较研究

分别采用饱和硫酸铵法和辛酸—硫酸铵法纯化兔抗酸性红73(acid red 73,AR73)免疫球蛋白G(IgG)。通过二喹啉甲酸(BCA)法、十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)试验、间接ELISA法分别测定IgG的浓度、纯度及效价。结果显示,饱和硫酸铵法所提IgG浓度为36.44 mg/mL,最高效价达1.6×10⁵;辛酸—硫酸铵法所提IgG浓度为15.79 mg/mL,最高效价达3.2×10⁵。提示,辛酸—硫酸铵法IgG纯度优于饱和硫酸铵法。     

聚乙二醇硫酸铵沉淀法对不同亚类单克隆抗体活性的影响

比较了以聚乙二醇和硫酸铵两种沉淀方法分离纯化腹水中单克隆抗体后,对不同亚类单克隆抗体活性的影响。结果表明,经３３％的硫酸铵沉淀腹水ＩｇＧ时,对其活性没有明显影响;沉淀ＩｇＭ时其活性明显降低。以聚乙二醇进行沉淀,对腹水单克隆抗体ＩｇＧ和ＩｇＭ活性均无明显影响,但依不同亚类的单克隆抗体,所选用的聚乙二醇浓度不同,对于ＩｇＧ,以１５％聚乙二醇沉淀较为合适,而沉淀ＩｇＭ时,则以６％聚乙二醇较为合适。     

Bone mineral response to ammonium sulphate offered as a lick supplement in beef calves

Sixteen Bonsmara calves (4 males, 12 females) between 10 and 18 months of age were blocked according to age and sex and randomly assigned to 2 groups. They were offered licks containing bone meal and salt (50:50 ratio) (control) and bone meal and ammonium sulphate (NH4SO4) at 1.25, 2.5, 5, 10, 15, and 18% (treatment) to evaluate the effects of dietary anions on bone phosphate (P) concentration. Bone P concentration was significantly (P < 0.05) higher in the NH4SO4 group compared with the control group, indicating that NH4SO4 was able to increase the P content of bone at each of the 6 concentrations used in the lick relative to the control animals, thereby improving the P status of the animals. Ammonium sulphate at 15% and 18 % in the lick also significantly (P < 0.05) increased bone P compared with the lower concentrations of NH4SO4. Bone calcium (Ca) fluctuated as a result of the acidogenic lick. There was absorption of Ca when P was being resorbed and resorption of Ca when P was being absorbed into and out of bone. Bone Ca:P ratio ranged from 3.2 to 6.4 among the control group and 1.6 to 4.3 among the treatment group. Animals receiving the acidogenic lick had a higher percentage ash compared to the control group for most of the experimental period. Bone magnesium (Mg) fluctuated in response to the acidogenic lick, and it was difficult to show a relationship between bone Mg and Ca or P. The overall mean cortical bone thickness was significantly (P < 0.05) greater in treatment (1.60 mm) compared with control (1.43 mm) calves and this was also true at sampling periods 2, 4, 5 and 6. Bone thickness followed bone P and not bone Ca. Results from this research indicate that the addition of ammonium sulphate to a lick had a beneficial effect in improving the P status by increasing bone P and improving the mineral status of bone by increasing the thickness of cortical bone and percentage ash.     

两种方法纯化猪血清免疫球蛋白IgG的比较

为了从猪血清中纯化免疫球蛋白IgG,得到纯度更高的样品,采用饱和硫酸铵分级沉淀和Protein G柱吸附分离两种方法进行纯化,将获得的IgG经SDS-PAGE凝胶电泳纯度鉴定和HPLC浓度测定,结果表明,利用Protein G柱吸附分离纯化出的样品在纯度和浓度方面都要高于盐析法,Protein G柱吸附分离纯化IgG更适合于实验室小规模的操作,能够获得纯度高的样品。     

小反刍兽疫免疫荧光诊断技术研究

为了建立小反刍兽疫(PPR)荧光抗体诊断方法,研究选用小反刍兽疫病毒(PPRV)Nigeria75/1减毒疫苗株制备抗原,免疫试验山羊,制备小反刍兽疫高免血清,用硫酸铵盐析法粗提免疫球蛋白(IgG),再应用提取的IgG制备异硫氰酸荧光素标记抗体。结果表明:所制备的荧光抗体对试验用Vero细胞组织抗原和犬瘟热病毒(CDV)抗原无反应性,对小反刍兽疫病毒感染细胞的检出敏感性为1×106稀释度。     

两种方法纯化免抗绵羊肺炎支原体IgG的比较

目的:比较两种方法纯化兔抗绵羊肺炎支原体IgG的效果。方法:采用饱和硫酸铵和辛酸-硫酸铵两种方法分离纯化兔抗绵羊肺炎支原体抗体,并用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)、分光光度计和琼脂扩散(AGP)试验对纯化产物进行相对分子质量、蛋白质含量及免疫活性进行鉴定。结果:纯化后抗体的蛋白含量基本相同;产物纯度和效价以辛酸-硫酸铵法较高。结论:辛酸硫酸铵法是一种简便、快速、高效的抗体纯化方法。     

Anti-A isoagglutinins in two blood type B cats are IgG and IgM

Sera from two blood type B cats had strong isoagglutinating and isohemolyzing titers against blood type A erythrocytes. In order to determine the class of the immunoglobulins, sera from the cats were pooled, ammonium sulfate precipitated, and gel filtered using sepharose 6B to separate the immunoglobulins by molecular size. The immunoglobulin concentrate separated into two fractions. The initial part of the first fraction was shown in an ELISA to contain IgM and to be devoid of IgG by immunoelectrophoresis and to have agglutinating activity (a titer of 1:4 with a protein concentration of 0.8 mg/ml). Treating this fraction with the reducing agent dithiothreitol (DTT) eliminated agglutinating activity. The latter portion of the second column fraction was shown to contain IgG by immunoelectrophoresis, and to be devoid of IgM by ELISA. Agglutinating activity was also present in the second fraction (a titer of 1:2 with a protein concentration of 1.9 mg/ml); hemagglutinating activity was not decreased by DTT treatment. These studies show that the predominant anti-A isoagglutinating activity in pooled sera from two blood type B cats is IgM and that some isoagglutinin activity can be associated with IgG.     

The effect of source of nitrogen on the dry matter yield,nitrogen and nitrate‐N content of Lolium multiflorum

Three sources of N fertilizer (urea, limestone ammonium nitrate (LAN) and ammonium sulphate) showed no consistent significant trends in terms of the dry matter yield and nitrogen or nitrate‐N content of midmar ryegrass. Ammonium sulphate was found to acidify the soil markedly, relative to urea, with LAN showing the least acidifying effect.     

A simple method for the isolation of immunoglobulin (Y) from the eggs of immunized hens

Hens were vaccinated with Salmonella typhi-murium and with serum-IgG from a goat. Antibodies (IgY) were isolated from the eggs by ammonium sulphate precipitation. The concentration of egg antibodies was equal or superior to the concentration of serum antibodies of the hen. The longlasting antibody plateau of 9 and 28 weeks, respectively, provides evidence of the high yields of specific IgY which can be collected from a single immunized hen.     

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。