纯化小鼠腹水中抗富含组氨酸糖蛋白单克隆抗体方法的比较

用羟基磷灰石柱怪析法，辛酸－硫酸铵沉淀法以及辛酸－硫酸铵沉淀法加羟基磷灰石桩层析法从小鼠腹水中提取纯化抗富含组氨酸糖蛋白单克隆抗体。结果表明，羟基磷灰石柱层析法简便、快速、其单克隆体产量和纯度均高，适合大多数实验室采用。     

用羟基磷灰石柱层析纯化抗人绒毛膜促性腺激素单克隆抗体(简报)

本文比较了用羟基磷灰石柱层析和DEAE-52纤维素两种方法从小鼠腹水液中纯化抗人绒毛膜促性腺激素单克隆抗体的结果。SDS-聚丙烯酰胺凝胶电泳图谱,紫外分光光度定量分析及放射免疫分析法检测抗体免疫活性的结果表明:(1)羟基磷灰石柱层析法得率高,未见有其它杂蛋白污染,纯化前后的免疫活     

长白白眉蝮蛇类凝血酶的研究Ⅰ：纯化方法的研究

采用四种纯化方法,对长白白眉蝮蛇类凝血酶的纯化途径进行研究。结果,阴阳离子交换柱、分子筛柱层析法周期较长,收率较低。羟基磷灰石柱法。对类凝血酶有较好的分离效果,但要求样品量小且纯度较高。亲和层析法分离类凝血酶,具有操作简便、纯化周期短、回收率高、处理样品量大等特点。     

高压液相色谱法纯化抗人大肠部单克隆抗体

采用Ｂｉｏ－Ｒａｄ公司的ＭＡＰＳ１００单克隆抗体纯化系统，利用羟基磷灰石层新 柱对鼠柱对复腹水中抗人大肠癌单克隆抗体进行分离纯化。结果：还原性ＳＤＳ－ＰＡＧＥ显示所获抗体为单一组分，ＥＬＩＳＡ证实抗体活性满意，所获抗体收率为２３％－５２％，重复进样收率稳定（３８．６３±２．２６）％，ｎ＝３。     

新生儿黄疸防治的实验研究

探讨人肝脏血红素加氧酶（ＨＯ）同功酶的性质，并应用血红素加氧酶抑制剂锡－原卟啉（ＳｎＰＰ）抑制酶的活性，为临床新生儿黄疸的防治提供新途径。方法采用２－二乙氨乙基－葡聚糖凝胶Ａ－２５和羟基磷灰石柱层析法从人肝脏分离纯化ＨＯ的同功酶，进行酶活性检测和十二烷基磺酸钠－聚丙烯酰胺凝胶电泳分析。结果人肝脏微粒体含ＨＯ的同功酶，按洗脱先后顺序分别得到分子量为３００００和３６０００的ＨＯ－１和ＨＯ－２。酶促反应中需相同辅酶参与，其中ＨＯ－１酶活性明显高于ＨＯ－２，两者之比为２．４１，从分子量和酶的催化活性分析，发现ＨＯ－１属诱导型的传统ＨＯ同功酶，ＨＯ－２为新发现的非诱导型ＨＯ的同功酶。结论ＳｎＰＰ能有效抑制ＨＯ－１的活性，使胆红素产生减少。     

pH为10至11时羟基磷灰石液相生成动力学

利用液相沉淀法研究了ｐＨ为１０－１１时，「Ｃａ＾２－」浓度大于０．５ｍｏｌ／Ｌ条件下基磷灰石的生成机理及其动力学。结果表明反应经历了从磷酸八钙很快转化为非晶态磷酸钙，继而转化为缺钙羟基磷酸石和羟基磷灰石的过程。     

大肠癌相关抗原CA－Hb_3的纯化及其在胃肠道肿瘤血清学诊断中的应用

用一株鼠抗人大肠癌单克险抗体Ｈｂ３，纯化后与溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ偶联制各免疫吸附柱。用免疫亲和层析法从大肠癌细胞株ＨＲＴ－１８细胞提取液中纯化相应抗原ＣＡ－Ｈｂ３。经多种实验证实ＣＡ－Ｈｂ３是一种新的大肠癌相关糖蛋白抗原、分子量约为６５Ｋｄ．以此纯化抗原作参考标准，用ＳａｎｄｗｉｃｈＥＬＩＳＡ方法检测大肠癌及消化迟其他肿瘤患者血清ＣＡ－Ｈｂ３水平。结果表明，ＣＡ－Ｈｂ３对胃肠运肿瘤诊断的敏感性和特异性较高，具有一定的临床价值。     

鼓槌石斛化学成分的研究

目的 研究鼓槌石斛的化学成分，为阐明其活性成分，开发其资源提供科学依据。方法 采用硅胶柱层析法进行分离，根据光谱数据鉴定结构。结果 从中分得4个化合物，分别为：2，4，7－三羟基－9，10－二氢菲（Ⅰ），2，7－二羟基－3，4，6－三甲氧基菲(Ⅱ），5，4′－二羟基－3，3′－二甲氧基联苄（Ⅲ），3，4－二羟基苯甲酸（Ⅳ）。结论 化合物Ⅰ-Ⅳ均为首次从石斛属植物中分得。     

DL-四氢巴马汀对大鼠单胺的排空作用

本文报道四季豆抗生育成分的提取和抗生育作用。提取采用丙酮沉淀法每100g 四季豆可提取10g。亦研究了硫酸铵分级盐析法,二乙氨乙基纤维素,二乙氨乙基葡聚糖凝胶柱层析和羟基磷灰石柱层析等方法。四季豆提取物经羟基磷灰石柱层析分离和纯化,可得4个洗脱峰,其中0.1M 磷酸缓冲液洗脱峰经醋酸纤维素薄膜电泳,可得一条主要区带和一条痕迹     

人骨形态形成蛋白的纯化及其诱导成骨的研究

本实验采用差示沉积、超滤及羟基磷灰石柱层析法,提取和纯化了人骨基质内骨形态形成蛋白,得到纯化的人骨形态形成蛋白,分子量为17000U(约17000道尔顿).以C_3H小鼠147只为实验动物,纯化的骨形态形成蛋白的诱导成骨率为100％。     

粒细胞集落刺激因子及其McAb的研究

利用B细胞杂交瘤技术,用rhG-CSF免疫BALB/c小鼠,将其脾细胞与SP2/0骨髓瘤细胞融合,成功地建立了3株能持续分泌特异抗rhG-CSF McAb的杂交瘤细胞。取其中2D_4-B_4杂交瘤细胞进行染色体数目测定,并将2D_4-B_4瘤细胞注入小鼠腹腔,获得抗体阳性腹水,测定提纯抗体IgG亚型、特异性、稳定性和亲和力,证实该McAb可以和hG-CSF及小鼠G-CSF特异结合。为进一步研究hG-CSF和人工合成G-CSF打下良好的基础。     

粒细胞集落剌激因子及其McAb的研究

Three hybridomas producing monoclonal antibodies (McAb) against recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been established by fusing mouse myeloma SP 2/0 cells with spleen cells from a BALB/c mouse immunized with rhG-CSF. Ascites was produced from BALB/c mice by injecting substrain 2D4-B4 hybridoma cells intraperitoneally. The antibody was purified either by the caprylic acid-ammonium sulfate method or by euglobulin precipitation. The caprylic acid-ammonium sulfate method was superior to euglobulin precipitation in terms of purification and recovery of IgG. The 2D4-B4 McAb was determined to belong to the IgG3 subclass with a molecular weight of about 172,400 It was proved by Western Blot to react specifically with rhG-CSF. The stability and affinity of the McAb were analyzed as well. The potential clinical and experimental applications of this McAb are discussed.     

Studies on monoclonal antibody against recombinant human granulocyte colony-stimulating factor.

Three hybridoma cell lines producing monoclonal antibodies (McAb) against recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been established by fusing mouse myeloma cell line SP2/0 cells with spleen cells from a BALB/c mouse immunized with rhG-CSF. Ascites was obtained from BALB/c mice by injecting the hybridoma cells intraperitoneally. Three McAbs were purified by the caprylic acid-ammonium sulfate method. For each, the IgG subclass, valence and activity, molecular weight, specificity and affinity were determined. The applications of McAbs against rhG-CSF in the clinic and laboratory are discussed.     

Studies on Purification of Methamidophos Monoclonal Antibodies and Comparative Immunoactivity of Purified Antibodies

Objective To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. Method Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linke dimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29μg/mL and 3.42μg/mL,respectively. Conelusion Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.     

羊布鲁菌BCSP31蛋白表达纯化及其单克隆抗体的制备

目的：表达并纯化羊布鲁菌BCSP31蛋白,制备其单克隆抗体（mAb）．方法：采用GST亲和层析纯化的方法纯化BCSP31蛋白．用纯化的BCSP31蛋白免疫小鼠,将小鼠脾细胞与骨髓瘤Sp2／0细胞融合,ELISA间接法筛选阳性克隆,3次克隆化后建立杂交瘤细胞系．采用小鼠腹腔接种杂交瘤细胞制备腹水,正辛酸-硫酸铵法纯化mAb．采用Western Blot和ELISA等方法鉴定mAb特性．结果：GST—BCSP31融合蛋白在25℃诱导时为可溶性表达,经亲和层析纯化,获得BCSP31纯化蛋白0．5g／L;Western Blot检测结果显示所获蛋白可与兔抗布鲁菌阳性血清特异性反应．获得2株mAb,分别命名为1F1,1E5．结论：BCSP31蛋白的纯化及其mAb的制备为布鲁菌病的诊断与防治奠定了一定的物质基础．     

Studies on Purification of Methamidophos Monoclonal Antibodies and Comoarative Immunoactivity of Purified Antibodies

Objective To purify Methamidophos (Met) monoclonal antibodies with two methods andcompare immune activity of purified antibodies. Method Caprylic acid ammonium sulphateprecipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method wereused to purify Met monoclonal antibodies, UV spectrum scanning was used to determine proteincontent and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linkedimmunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mLand 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASPmethod. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was181.26 μg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01μg/mL and 1.03 μg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82μg/mL, and the linear working range and LOD were 10.91-11412.29 ug/mL and 3.42 μg/mL,respectively. Conclusion Antibodies purified by SPA method are better than those by CAASPmethod, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelledtesting paper for analyzing Met residue in vegetable and drink water.     

小鼠脑Ca~(2+)/CaM依赖性蛋白激酶Ⅱ的鉴定及苄普地尔对其酶活力的抑制作用

用DE_(52)、磷酸纤维素和Sephaery S—300柱层析从小白鼠脑中提纯了Ca~(2+)/CaM依赖性蛋白激酶Ⅱ并进行了鉴定。凝胶过滤法测定全酶分子量为55万,在SDS-PAGE中显示出两条区带,其亚基分子量分别为5万和5.7万。该酶活力依赖于Ca~(2+)和CaM,AC_(50)分别为28μmol/L和2.2μmol/L。苄普地尔对酶有抑制作用,IC_(50)约为250μmol/L。     

重组登革病毒2型NS1蛋白的分泌表达及其抗体制备

目的 分泌表达重组登革病毒2型NS1蛋白,并制备兔抗NS1多克隆抗体.方法 应用毕赤酵母表达系统表达全长登革病毒2型NS1蛋白,制备抗原,免疫家兔;采集免疫血清,制备兔抗NS1蛋白多克隆抗体;应用Western-blot、ELISA法鉴定和检测抗体效价;经辛酸-硫酸铵法、亲和层析法纯化抗体,SDS-PAGE电泳检测抗体的纯度;用Western-blot、ELISA法检测纯化后IsG性质及效价.结果 重组NS1蛋白获得分泌表达,其免疫血清经Western-blot、ELISA法证实获得特异性兔抗NS1多克隆抗体,抗体效价为1:6000.结论 重组登革病毒2型NS1蛋白在毕赤酵母真核表达系统中高效表达,纯化产物有较强的免疫原性,成功获得特异性兔抗NS1多克隆抗体,为进一步研究登革病毒NS1蛋白及其抗体在登革病毒致病与免疫机制中的作用奠定了基础.     

鼠源性抗人IgMμ链单克隆抗体的制备和鉴定

目的 :建立能稳定分泌高效价抗人 Ig Mμ链单克隆抗体的细胞株 ,收获并纯化抗体用于进一步研究。方法 :小鼠免疫脾细胞与骨髓瘤细胞用 PEG融合 ,小鼠体内诱生腹水的方法收获大量单克隆抗体 ,辛酸硫酸铵提纯 ,并鉴定其纯度。结果 :获得 3个稳定分泌抗体的细胞株 M1、M2、M3,鉴定其亚类分别为 Ig G3、Ig G2 a、Ig G1;腹水抗体纯化后获得纯化抗体含量分别为 0 .36 mg/ ml、0 .2 8mg/ ml、3.71mg/ ml。结论 :用 PEG进行细胞融合 ,辛酸硫酸铵盐析法纯化能够获得较高效价和纯度的抗体     

LpB：E标准物的制备

用纯化的人ＬＤＬ（ａｐｏＢ）免疫山羊，获得特异性羊抗人ａｐｏＢ抗血清。将用正辛酸、硫酸铵沉淀和Ｐｒｏｔｅｉｎ－Ｇ亲和层析纯化的羊抗人ａｐｏＢＩｇＧ与ＣＮＢｒ－ａｃｔｉｖａｔｅｄＳｅｐｈａｒｏｓｅ４Ｂ共价交联，可制备获得特异性抗体亲和层析柱。将适量的人血清通过该亲和柱，用高ｐＨ缓冲液冲洗后，用低ｐＨ缓冲液洗脱，可获得含ａｐｏＢ的脂蛋白，其中包括了同时含ａｐｏＢ和ａｐｏＥ的ＬｐＢ：Ｅ组分。测定其ａｐｏＥ含量后，该组分可作为测定ＬｐＢ：Ｅ的标准物。