A蛋白亲和色谱法纯化动物血清抗体的效果比较

比较Hitrap A蛋白琼脂糖凝胶亲和色谱系统,对不同动物血清或抗血清抗体的纯化效率,提供选择抗体纯化方法的依据。该方法简使快速,纯化的抗体纯度高,能较好地保持抗体免疫学活性。色谱柱反复使用40多次,纯化抗体效率不变。但A蛋白对不同动物血清的IgG吸附能力不同,对兔和豚鼠的血清抗体吸附能力强,而对小鼠、山羊和驴的血清抗体吸附能力较弱。说明虽然该方法纯化的抗体纯度高,但它对动物品种有选择。因此,应根据动物品种,选择适合的纯化方法。     

G蛋白亲和色谱法纯化动物血清、抗体效果比较

比较 Hitrap G蛋白 Sepharose亲和色谱系统对不同动物血清或腹水的纯化效果 ,为抗体纯化提供依据。结果显示 ,G蛋白对不同动物血清 Ig G吸附能力不同 ,体现在单位体积抗体回收量明显不同 ,这一特点与 A蛋白亲和色谱系统相似。该方法简便快速 ,纯化抗体纯度高 ,免疫活性好 ,色谱柱可反复使用多次     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

重组人尿激酶原的分离纯化及性质研究

本文报道从人尿激酶原（ｐｒｏ－ｕｒｏｋｉｎａｓｅ,ｐｒｏ－ＵＫ）基因重组工程菌Ｅ．ｃｏｌｉＪＡ２２１表达产物的复性液中纯化尿激酶原的方法。复性产物经Ｚｎ＾２＋选择性沉淀,尿激酶抗体亲和柱层析,ｂｅｎｚａｍｉｄｉｎｅ－Ｓｅｐｈａｒｏｓｅ ＣＬ ４Ｂ亲和吸附,即得比活达１１００００ＩＵ／ｍｇ的纯化产品。样品经ＳＤＳ－ＰＡＧＥ鉴定,在还原及非还原条件下,均表现为分子量为４３ｋｄ的单一条带。动力学研究测得     

九种重要经济动物二级抗体的制备和HRP标记

目的制备兔抗9种重要经济动物的二级抗体,并进行辣根过氧化酶标记,为经济动物血清学检测系统的建立提供工具。方法利用饱和硫酸铵沉淀粗纯抗体后,再利用protein A或protein G亲和层析的方法进一步纯化动物血清IgG,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用Protein A亲和层析的方法纯化兔抗经济动物的二级抗体,采用简易过碘酸钠法对纯化的二级抗体进行HRP标记,通过ELISA方法测定标记抗体的效价,并利用Western blotting考察标记抗体的特异性。结果纯化了家猪,绵羊、山羊、牛、马、驴、狐狸、貉和黑貂9种重要经济动物的血清IgG,免疫双扩散法测定兔抗血清效价均达到1：32,并对纯化的兔抗家猪,绵羊、山羊、牛、马、驴、狐狸、貉和黑貂9种重要经济动物二级抗体进行了HRP标记,ELISA测定标记抗体的效价达到1：（2000～15000）左右,Western blots显示标记抗体具有很好的特异性。结论成功制备了HRP标记的兔抗9种重要经济动物的二级抗体,为经济动物血清学检测体系的建立提供了工具。     

雄激素受体融合蛋白的表达及其多克隆抗体的制备与纯化

将雄激素受体的ｃＤＮＡ片段克隆到表达质粒ｐＧＥＸ－３Ｘ内,在Ｅ．ｃｏｌｉ中表达,得到可溶性表达产物ＧＳＴ－ＡＲ。用该融合蛋白制得的兔多克隆抗体,经ＧＳＴ－Ｓｅｐｈａｒｏｓｅ－４Ｂ亲和吸附纯化后,表现出较好的抗ＡＲ的专一性,可用于ＡＲ结构和功能的研究。     

尿激酶型纤溶酶原激活剂单克隆抗体的生产及其在抗原纯化中的应用

从ＣＨＯ工程细胞培养上清初步纯化的ｕＰＡ免疫ＢＡＬＢ／ｃ小鼠,通过杂交瘤技术制备单克隆抗体细胞株,取其中一株３８－１－７株作高密度大量培养,细胞密度达１３．２×１０６／ｍＬ时,抗体滴度为１∶６１．４４×１０４。用自制的ｕＰＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱纯化抗体。抗体滴度提高２４３倍。纯化后的抗体与活化的Ｓｅｐｈａｒｏｓｅ４Ｂ珠交联,制成ＩｇＧ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析抗体柱,亲和力常数：１．２８×１０９（ｍｏｌ／Ｌ）－１,交联率：８３．５％。直接从培养上清纯化ｕＰＡ,纯度为９６．３％,回收率：８１．６％±１９,纯化倍数：５０倍左右,比活１．１１±０．２９×１０５。试验结果表明该法效果好,方法简单、操作方便、值得进一步研究和应用。     

丙型肝炎病毒基因组部份NS5区蛋白基因在大肠杆菌中的表达

在大肠杆菌中表达了丙型肝炎病毒基因组ＮＳ５区Ａ段和部份Ｂ段蛋白。ＮＳ５Ａ蛋白溶于水,可经过ＧＳＴ亲和层析柱纯化;ＮＳ５Ｂ蛋白不溶于水,经ＰＢＳ－Ｔｒｉｔｏｎ Ｘ－１００洗涤,尿素溶解后,通过离子交换柱纯化。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｍｂｌｏｔ分析,ＮＳ５Ａ蛋白除在５７ｋＤ左右有条带外,还有不同程度的降解产物;ＮＳ５Ｂ蛋白主要在５８ｋＤ左右有条带出现。为查明表达蛋白抗体在病人血清中的分布,取９     

重组轮状病毒SA11 Vp4抗原表位诱导广泛交叉中和抗体反应

本文报导在ＲＨＶ－ＲＮＡ２载体系统中表达的轮状病毒（ＲＶ）Ｖｐ４胰酶切割位点区抗原位可诱导动物产生具有广泛交叉中和反应的血清抗体。携带有抗原表位ＲＥＡ、ＲＥＢ和ＲＥＣ,及其组合ＲＥＡ＋ＲＥＢ＋ＲＥＣ基因的ｐＥＴ重组质粒,高水平表达了这些与载体蛋白嵌合的抗原表位。进一步研究结果表明,这些抗原表位所诱导的动物血清抗体,具有广泛识别同源及异源血清型ＲＶ抗原的能力,和体外中和这些同源及异源血清型ＲＶ病毒感     

影响PVDF膜对蛋白吸附效率的主要因素

通过结合有抗原的PVDF膜进行亲和纯化是小规模快速获取特异性抗体的一种重要手段.该方法的关键步骤之一是将抗原蛋白有效地吸附到PVDF膜上.尽管人们广泛使用PVDF膜亲和纯化抗体,但对影响PVDF膜吸附抗原蛋白的因素至今仍缺乏系统研究.以BSA为模型蛋白,检测在不同蛋白浓度、不同孵育时间、不同溶质溶液和不同pH值的情况下,PVDF膜吸附蛋白的效率.当蛋白溶液为PBS时,1 cm2 PVDF膜与2 mg·mLl BSA溶液孵育1h可达到其最大吸附率.常见的蛋白质缓冲液组分对PVDF膜的蛋白吸附率影响不一.其中,咪唑、Tris-HCl、NaCl、KCl在广泛的浓度范围内对PVDF膜吸附蛋白没有明显影响,而较高浓度的SDS、Triton-X 100、Tween-20、乙二醇、甘油、盐酸胍、尿素等可显著降低PVDF膜对蛋白的吸附效率,在pH为6.5～9.5的Tris-HCl中,随着pH的增高而吸附效率降低.     

貉血清IgG纯化及抗血清的制备

目的 制备高纯度貉血清IgG和兔抗貉IgG抗血清,作为建立多种动物抗体检测技术的储备。方法 采用Hitrap Protein A亲和层析及盐析再沉淀法纯化貉血清IgG,通过PAGE电泳和Western-Blot免疫印迹法对IgG作纯度及免疫活性检测;常规免疫法制备兔抗貉IgG血清。结果 貉血清IgG与Protein G虽有较强的结合力,但同时也结合血清中其他杂蛋白;用二步纯化法可从5 mL貉血清中纯化IgG约7 mg,电泳和免疫印迹测定显示,IgG纯度大于95%,常规免疫法制备抗血清免疫双扩散效价达1∶32。结论 建立了可行的貉血清IgG的纯化方法和高效价的兔抗貉血清IgG抗血清,为貉血清IgG二级抗体酶联物的制备储备了资源。     

虎血清免疫球蛋白（IgG）的纯化及纯度、活性鉴定

目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。     

两种果子狸血清IgG纯化方法 的比较

目的 探讨一种具有简便快捷、高纯度、活性好的果子狸血清IgG纯化方法。方法 比较Hitrap Protein A亲和层析和PAGE电泳两种纯化法对果子狸血清IgG的纯化,用PAGE还原电泳和Western-Blot法对IgG作纯度鉴定。结果 对Hitrap Protein A纯化的果子狸血清IgG,其活性虽好,但纯度不高;而非还原PAGE电泳所纯化的果子狸血清IgG不但纯度高（〉95%）、并具有较强的免疫活性。结论 非还原PAGE电泳法纯化果子狸血清IgG,是一种具有高纯度、免疫活性强的纯化方法 。     

Immunochemical characterization of an IgG-binding protein of Streptococcus suis

Several bacterial species express surface proteins with affinity for the constant region (F_c) of immunoglobulin (Ig) of different animal species. Previous studies from our group have reported the presence of an IgG-binding protein in various serotypes of Streptococcus suis . This molecule was also shown to bind in a non-immune fashion chicken IgY and to our knowledge this characteristic is unique. In the present study, by dot-blotting, we showed that the native protein, obtained by affinity chromatography, reacted more strongly with IgG from various animal species than the denatured material. Using a competitive enzyme-linked immunosorbent assay the affinity of the native 60-kDa protein (previously identified as a 52-kDa protein) towards IgG of various animal species was compared to pig IgG. Bovine, goat and human IgG were able to compete effectively with pig IgG whereas chicken IgY constituted a poor competitor. Peptide mapping analysis using denatured protein indicated that pig and bovine IgG recognized the same proteolytic fragment whereas chicken IgY did not. The smallest proteolytic fragment that retained the binding activity towards the IgG of the different animal species tested had a molecular mass of approximately 40 kDa. Fragments with M _r<40 kDa showed specific binding activities. That is, the smallest fragment binding pig and bovine IgG had a M _r of 30 kDa whereas for goat and human IgG a fragment of less than 16 kDa still showed binding activity. Finally, we observed that antisera raised against a heat-shock protein of Pseudomonas aeruginosa reacted with the 60-kDa S. suis protein indicating that the S. suis 60-kDa protein is a member of the 60-kDa hsp family that possesses the characteristic of binding in a non-immune way mammalian IgG and chicken IgY.     

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.     

The production of specific immunoenzyme conjugates to human immunoglobulins by the glutaraldehyde method]

The results of studies aimed at obtaining class-specific conjugates to human immunoglobulins to be used in the enzyme immunoassay (EIA) are presented. At the first stage of the studies purified IgA, IgM and IgG preparations were obtained. These preparations were used for obtaining immunologically active immunosorbents on the basis of bromocyanic Sepharose. Specific antibodies to human IgA, IgM and IgG were isolated from animal sera by the method of affinity chromatography. These antibodies were conjugated with peroxidase by the glutaraldehyde method. The specific activity of the conjugates were determined in EIA. The results thus obtained revealed that all preparations exhibited high specific activity and gave no cross reactions with immunoglobulins of other classes.     

Control of allergic reactivity in human filariasis. Predominant localization of blocking antibody to the IgG4 subclass.

Patients with chronic helminth infections, despite having abundant basophils and mast cells specifically sensitized with antiparasite IgE and often exposed repeatedly to parasite Ag, rarely manifest allergic symptoms. This control of clinical allergic reactivity likely results from Ag-specific IgG "blocking antibodies" shown previously to be abundant in the sera of such patients. In the present study we used two approaches to determine in which of the four IgG subclasses this blocking activity was localized. First, specific antifilarial antibodies of each of the four IgG subclasses were quantified in the sera of 28 patients with Bancroftian filariasis and correlated with the levels of blocking activity in these sera (determined by histamine release assays). A significant correlation with blocking activity was seen only for antibodies of the IgG4 subclass, and, indeed, the correlation was especially strong in the group of totally asymptomatic patients (but with microfilariae circulating in the blood) in whom blocking antibody levels were highest. Interestingly, however, if the analysis excluded these asymptomatic microfilaremic patients and focused instead on those with lymphatic inflammatory pathology (who had relatively low levels of both serum blocking activity and specific IgG4 antibodies), then the small amount of blocking activity found in these sera correlated only with the levels of IgG1 subclass antibodies. The second approach utilized selective depletion of IgG4 (by anti-IgG4 affinity columns) from the sera of three microfilaremic patients with high levels of blocking activity and demonstrated clearly that removal of IgG4 abolished the majority of the blocking activity in these sera (53, 78, and 81%). These two sets of findings demonstrate a predominant role for specific IgG4 antibodies in blocking IgE-mediated allergic responses to the parasite Ag in vitro, but they also indicate that in some situations IgG1 antibodies can block such reactions. Furthermore, the correlation demonstrated between patients' clinical presentations and the levels of both their specific IgG4 antibodies and serum blocking activity suggests that these antibodies play a similar role in vivo as well.     

Molecular studies of rheumatoid factor using pseudobioaffinity membrane chromatography

Using pseudobioaffinity ligand L-histidine immobilized to poly(ethylene vinyl alcohol) hollow fiber membrane is an interesting approach for the purification of total IgG and its subclasses from untreated serum in a single step. Gentle adsorption and elution conditions of this chromatography system allow efficient recovery of the protein in its native form. This approach was employed for the recovery and molecular study of rheumatoid factor (RF), an anti-IgG autoantibody (AAb) that form immune complexes with autologous IgG Abs in the sera. The purity of the recovered molecule was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), revealed a 150-kDa IgG band and an additional approximately 300 kDa band which may be RF bound IgG complex. Since RF is an AAb, the purified protein was studied for its catalytic functions like peptide, DNA, and RNA hydrolyzing activities. The substrate Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) hydrolyzing activity by total IgG from different patient sera was found to be greater than healthy controls. In an effort to identify the subclass specificity for the proteolytic function, the pre-purified total IgG fractions from rheumatoid arthritis (RA) sera were subjected to rechromatography using a discriminating buffer. In this experiment, the activity was found in the non-retained fractions suggesting IgG2 specificity for the catalytic function. A comparative study between different catalytic functions was performed for IgG separated from individual patient.