抗汉坦病毒等三种单克隆抗体细胞株的建立及分析应用

以纯化的病毒抗原加完全福氏佐剂,免疫BALB/c小鼠,再利用杂交瘤技术建立针对汉坦病毒、狂犬病毒及乙型脑炎病毒的单克隆抗体(McAb)细胞株,制备并提纯标记McAb,应用于各种实验和检测工作。结果获得了6株分泌抗汉坦病毒McAb杂交瘤细胞株、6株分泌抗狂犬病毒McAb杂交瘤细胞株、2株分泌抗乙型脑炎病毒McAb杂交瘤细胞株,共14株,并对它们的特性进行了分析。各McAb效价不尽相同,有的高达10-7,有的则不足10-3。各McAb亚型以IgG型为主,少数为IgM型;轻链以κ型为主,个别为λ型或阴性。McAb与纯化的病毒抗原有明显的特异性吸附(P<0.01)。有的McAb有相同的作用位点,有的McAb则没有,但与其它McAb有交叉反应。通过对McAb进行提纯和标记,建立了双抗体夹心ELISA法,用于病毒抗原的检测。实验表明获得的McAb有较好的生物学特性,可与相应的病毒抗原特异性结合,用于免疫学检测及其它多种用途。     

酶标记抗人IgA单克隆抗体及其在Epstein-Barr病毒免疫酶技术中的应用

继成功地建立了4株分泌高效价抗人IgA McAb杂交瘤细胞株后,我们将制备出的McAb用于改进鼻咽癌早期诊断中的IgA/VCA和IgA/EA免疫酶技术,并探索了高效价抗人IgA McAb的纯化、辣根过氧化物酶(HRP)标记及有效保存的方法,结果令人满意。 采用饱和硫酸铵沉淀法及Sephadex A-50吸附法对来自细胞培养上清液和腹水的     

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.     

用斑点酶联免疫吸附试验检测轮状病毒抗原

我们用国产试剂和材料建立了一种以硝酸纤维素膜(NCM)作为固相载体的斑点酶联免疫吸附试验(Dot-ELISA),用于检测粪标本中的轮状病毒. 首先用铁笔将NCM划成5×5mm的小方格,然后将免抗人轮状病毒IgG打点至方格中心,室温凉干后,置封闭液中室温作用1小时,取出用PBST洗涤3～5次,每次5分钟,然后将膜直接浸入待检粪样、阳性抗原对照和阴性抗原对照中.置37℃ 30～60分钟,洗涤。随后将上述诊断膜浸入酶标兔抗人轮状病毒IgG中,室温45～60分钟,洗涤。用底物DAB显色数分钟,至阳性对照样本出现明显褐色斑点时,用自来水终止反应。根据斑点颜色深浅,肉眼即可判断阳性或阴性结果。     

癌胚抗原单克隆抗体的制备及免疫学性状的研究

用由结肠癌肝转移灶纯化的cEA抗原免疫BALB／c小鼠,其脾细胞与鼠骨髓瘤细胞sP2／0融合,制备产生cEAMcAb的杂交瘤。用ELIsA法检测与c已A,NcA、NcA一2的反应特异性。获得8株仅与cEA有结合反应的McAb,通过阻断抑制试验证明这8株McAb认识cEA分子上三个不同的抗原决定簇。它们的免疫球蛋白亚类同是IgG-．     

人心肌肌钙蛋白Ⅰ单克隆抗体及多克隆抗体的制备

目的:以重组人心肌肌钙蛋白Ⅰ(cTnⅠ)为抗原制备鼠源单克隆抗体(McAb)及兔源多克隆抗体,并鉴定抗体的特性。方法:以纯化的重组人cTnⅠ为抗原免疫BALB/c小鼠,取鼠脾细胞同Sp2/0骨髓瘤细胞融合,利用选择培养基筛选融合的杂交瘤细胞,用有限稀释法分离获得能够稳定分泌抗cTnⅠ的McAb阳性克隆,并利用体内诱生法大规模制备McAb,用辛酸-硫酸铵沉淀法纯化抗体;兔多抗制备以cTnⅠ为抗原常规免疫后取其血清;用间接ELISA和Western印迹鉴定抗体的性质。结果:经ELISA鉴定,筛选出5株能分泌cTnⅠMcAb的杂交瘤细胞株,即C5B2、C5B3、C5B4、C5B1、B1A6,效价最高的B1A6株分泌的McAb为IgG3型,纯化后效价为1∶10000,亲和常数为1.08×10-9mol/L,Western印迹鉴定表明cTnⅠMcAb有良好的特异性;兔多抗纯化后的效价为1∶8000。结论:制备了具有良好特性的cTnⅠMcAb和多克隆抗体。     

幽门螺杆菌尿素酶单克隆抗体的研制及鉴定

应用杂交瘤技术 ,建立稳定分泌抗幽门螺杆菌尿素酶单克隆抗体 (HPU McAb)的细胞系。用纯化幽门螺杆菌尿素酶 (HPU)抗原加福氏佐剂免疫BALB/c小鼠 ,按常规方法进行细胞融合 ,以纯化HPU抗原包被 ,间接ELISA方法筛选 ,并经多次有限稀释法克隆。获得 6株抗HPU的杂交瘤 ,腹水效价达 1∶6 .4× 10 4～ 1∶2 .5 6× 10 5,特异性专一 ,并对其进行体内外连续传代 3个月 ,分泌抗体能力稳定。IgG亚类分型主要为IgG1型。该细胞系能稳定分泌幽门螺杆菌尿素酶单抗。     

用单克隆抗体ELISA研究和分析霍乱弧菌抗原结果

本文用ELISA以12株单克隆抗体(McAb)对55株EI Tor弧菌和用遗传学方法突变的28株霍乱弧菌的菌体抗原进行了研究,同时与四种常规分型方法进行对比试验。用12株McAb可将55株El Tor弧菌分成11种抗原决定簇和18个McAb型,将遗传学方法的突变株分成9种抗原决定簇和10个McAb型。其中一株McAb2B_1H_1F_3能识别所有55株EI Tor弧菌和大部分突变株霍乱弧菌,表明有种的特异性;其余11株McAb与各型霍乱弧菌的抗原反应,均有差异,与这些菌株的反应百分比各不相同(21.0％～100％),表明可能存在着亚型和亚群。文中着重讨论了用McAb研究分析抗原的重要意义和McAb用于诊断的价值。     

结核分枝杆菌38kDa-U1融合抗原的表达、纯化及其抗原活性测定

由于结核病人抗体反应存在异质性,把单一抗原用于血清学诊断存在阳性率较低的缺陷。为获得适于结核病临床诊断用抗原,本研究将结核分枝杆菌38 ku抗原和尿蛋白(urine protein 1,U1)进行融合表达、纯化并测定其抗原活性。用基因拼接法将结核分枝杆菌编码38 ku抗原和U1蛋白的基因拼接在一起,克隆到pMD18-T上,进一步构建重组质料pET28a-38kDa-U1,并转化E.coliBL21,通过IPTG诱导实现其高效表达,采用亲和层析纯化表达产物。用纯化的融合抗原免疫家兔获得抗血清,用该血清以及活动性肺结核病人血清对纯化产物的抗原活性进行测定。结果成功构…     

玉米细菌性枯萎病菌改良Dot-ELISA检测研究

通过激光切割技术加工出硝酸纤维素膜小圆片并粘贴在已打孔的塑料胶条上,制成包含8个圆片的NCM条(NCM test strip),进一步在NCM条上建立了玉米细菌性枯萎病菌的Dot-ELISA检测方法.研究发现,在NCM圆片上的Dot-ELISA检测灵敏度与点样量密切相关,采用10 μL/点的加样量比通常的1 μL/点可提高检测灵敏度10-100倍;间接Dot-ELISA的检测灵敏度是双抗夹心dot-ELISA的10倍,该结果进一步在微孔板ELISA的检测中得到证实.玉米细菌性枯萎病菌的改良Dot-ELISA检测是一种较为灵敏、快速、稳定、规范的实验方法,为进一步研究该病菌的微流控芯片斑点免疫检测方法奠定了前期工作基础.     

Purification of human chorionic gonadotrophin from urine by membrane filtration affinity chromatography with a positively charged membrane.

A new method of affinity chromatography, termed membrane filtration affinity chromatography (MFAC), has been developed and applied to purify HCG from urine. By filtrating urine through ZBM (HCG in urine would bind to the antibody on ZBM) and by dissociating the HCG from the antibody on ZBM in purified form, we developed the MFAC and purified HCG from urine of pregnant women by MFAC. The purified HCG showed a single band in polyacrylamide gel electrophoresis. ZBM (1 cm(2)) could harvest 90.3 microg HCG, which showed immunoactivity of 8554 IU/mg. The rate of recovery was 87%. CONCLUSION: MFAC with ZBM is an effective method, which is much easier and cheaper than conventional affinity chromatography for purification of proteins from solution, especially from highly diluted solution.     

抗胶质瘤细胞单克隆抗体SZ-38及其识别抗原的生化特征

本文以人脑胶质瘤细胞系SHG-44为抗原,利用杂交瘤技术建立了一株恒定地分泌抗胶质瘤细胞单克隆抗体(McAb)的杂交瘤细胞株SZ-38。McAb SZ-38与9/10胶质瘤细胞系发生强结合反应。而与淋巴细胞、ABO型红细胞等所有正常血液细胞及绝大多数被检测肿瘤细胞系无反应。经免疫转移电泳及免疫沉淀法鉴定,该McAb识别抗原为胶质瘤细胞膜上Mw47,000糖蛋白。应用SPA-Sepharose 4B提纯MeAb SZ-38,再把纯化抗体交联于Sepharose 4B,以McAb亲和层析法提取SZ-38抗原,经SDS-PAGE证实其有较高的纯度。     

猪生长激素及其单克隆抗体的制备与纯化

本文介绍一种简便快捷提纯猪生长激素的方法。通过杂交瘤技术,首次获得抗猪生长激素的单克隆抗体,并讨论和比较从腹水纯化单克隆抗体的各种方法。     

草鱼生长激素单抗免疫亲和柱的制备及初步应用

用纯化的草鱼生长激素单克隆抗体偶联到CNBr活化的Sepharose4B凝胶上,制成约10ml的亲和层析柱．用该柱一步纯化了重组鲤鱼生长激素基因的表达产物．偶联有13．65mg单克隆抗体的亲和柱一次可纯化得到约0．7mg重组鲤鱼生长激素．酶联免疫受体测定表明它具有强烈的生物学活性,SDS－PAGE表明它为单一蛋白带,分子量约为22000．     

抗EHFV的独特型抗体在小鼠体内诱导免疫应答的研究

本文介绍用流行性出血热（EHF）病毒J10株制备单克隆抗体（McAb）,以及用7个McAb免疫家兔,使其产生抗EHF病毒McAb的抗体即抗独特型 抗体（Ab2).Ab2能在体外同出血热病人恢复期血清和EHF病毒免疫的兔血清发生特异性结合。再经EHF－McAb亲和层析法分离提纯Ab2,免疫BALb/c小鼠,将所获得的免疫血清（Ab3）用荧光和ELISA分别加以测定。结果表明,抗－抗独特型抗体可在体外识别EHF病毒,而不能同病病人恢复期血清发生结合,从而支持免疫网络学说,可能为我们提供一种新型的抗原来源途径。     

Purification of the protein X of Streptococcus agalactiae with a monoclonal antibody

The protein X of Steptococcus agalactiae is a surface antigen included in the typing scheme of group B streptococci (GBS). We have developed a monoclonal antibody to the protein X and used it to purify this antigen by affinity chromatography. Electrophoresis in polyacrylamide, and immunoblotting using the monoclonal antibody or a rabbit antiserum raised with the affinity purified protein X, revealed a major band in the region of 200 kDa and a smaller one at 100 kDa. The isolated protein X will make possible investigations of its potential role in virulence and protection.     

hGH单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立

采用杂交瘤法制备单克隆抗体,并用辛酸-硫酸铵法纯化单抗,通过ELISA方法和Western blotting测定抗体的效价与特异性,并进行抗体类型、相对亲和力测定;应用纯化的单抗建立hGH双抗体夹心ELISA检测方法。筛选出两株可以稳定分泌抗hGH单抗的杂交瘤细胞株,分别命名为3E11、2G9,抗体类型均为IgG1,抗体滴度均可达10-10,特异性好,相对亲和力高,以筛选到的两株单抗建立的双抗夹心ELISA法线性范围为0.09～1.5625ng/mL,R2>0.9,灵敏度为0.09ng/mL。筛选出高效抗hGH的单抗,并建立了hGH双抗体夹心ELISA检测方法。     

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

Screening for weak monoclonal antibodies in hybridoma technology

As the interest in weak-affinity antibodies has been widened by their introduction to various analytical techniques such as HPLC, capillary electrophoresis and biosensors, there has been a need for new screening/monitoring methods. In this study, weak-affinity chromatography was adopted to screen/monitor directly for monoclonal antibodies in ascites. Monoclonal antibodies against a carbohydrate antigen (maltohexaose) were used to evaluate this approach. In short, malthohexaose was immobilized on an HPLC support in such a configuration to allow, during HPLC, retardation of weak monoclonal antibodies. Based on the retention, the affinity or the avidity, as determined by the presence of multiple binding of the monoclonal antibody towards antigen, can be estimated. In this way it is possible to select clones of hybridomas that produce desired weak monoclonal antibodies. Adjustments in temperature (10-20 degrees C) were used to moderate the retention and hence affinity of the weak monoclonal antibodies during chromatography.     

小鼠腹水型肝癌H22a细胞浆胞内磷蛋白磷酸酶的纯化和性质

 磷蛋白磷酸酶是磷酸化/脱磷酸化作用中重要的调节酶。本文建立了小鼠腹水型肝癌细胞胞浆内磷蛋白磷酸酶(PrP)的纯化方法。用~(32)P-酪蛋白为底物测定活力。经纯化的酶纯度提高1380倍,聚丙烯酰胺梯度凝胶电泳检查,只有一条泳带。用凝胶过滤法和聚丙烯酰胺梯度凝胶电泳法测得该酶分子量为200,000。该酶催化~(32)P-酪蛋白脱磷酸化反应的最适pH7.2,对热不稳定。