对氧磷单克隆抗体的研制及鉴定

重氮化法使对氧磷（Ｅ６００）与牛血清白蛋白（ＢＳＡ）、血蓝蛋白偶合合成人工抗原。用人工抗原Ｅ６００－ ＢＳＡ免疫Ｂａｌｂ／ｃ小鼠,然后将ＳＰ２／０小鼠骨髓瘤细胞和Ｅ６００－ ＢＳＡ免疫的Ｂａｌｂ／ｃ小鼠脾脏细胞融合,成功的建立了能分泌抗对氧磷单克隆抗体的杂交瘤细胞株。并分析了该细胞株的染色体及对其产生的单克隆抗体的类型、特异性、亲和力进行了鉴定和检测。上述杂交瘤细胞连续传代或冻存１０周,其分泌抗体水平稳定     

甲基对硫磷单克隆抗体的研制及鉴定

目的研制甲基对硫磷(M1605)单克隆抗体(McAb).方法人工抗原M1605-TTH免疫BALB/C小鼠,用被免疫的脾脏淋巴细胞与骨髓瘤细胞SP2/0在融合剂PEG4000作用下融合.经过HAT培养液选择性培养、间接ELISA法筛选和有限稀释克隆后,鉴定该杂交瘤细胞株和McAb.结果获得2株稳定分泌McAb的杂交瘤细胞株G12、H02,其培养液和腹水滴度为1103和1105.进一步研究G12发现,该杂交瘤细胞株的染色体数为99～108,其McAb分类为IgM,该McAb的亲和力常数为1.67×105L/mol,且具有较好的特异性.结论获得抗M1605的特异性McAb,为建立M1605简便、快速、特异的生物检测方法提供了必要条件.     

抗河豚毒素单克隆抗体的制备及其特性的初步研究

将用合成的匙孔形虫戚血蓝蛋白－甲醛－河豚毒素（ＫＬＨ－ＨＣＨＯ－ＴＴＸ）连接物免疫的ＢＡＬＢ／ｃ小鼠的脾细胞与小鼠骨髓瘤细胞系Ｓｐ２／０融合，经３～４次亚克隆，建立了３个稳定分泌抗河豚毒素的杂交瘤细胞株，分别命名为１Ｇ３、４Ｇ３、６Ｄ９。其中１Ｇ３和４Ｇ３分属ＩｇＧ２ｂ亚类，６Ｄ９属ＩｇＧ１亚类。腹水的抗体稀释度为１∶１０５～１：５×１０６。用竞争抑制性酶联免疫吸附试验（ＥＬＩＳＡ）测定三种抗体的最低反应浓度为１×１０－３ｍｇ／Ｌ。     

金黄色葡萄球菌肠毒素单克隆抗体制备与应用

目的制备抗B型金黄色葡萄球菌肠毒素单克隆抗体。方法纯化B型金黄色葡萄球菌肠毒素(SEB)免疫BALB／C小鼠的脾细胞与SO2／0骨髓瘤细胞融合，经筛选及克隆化，共获得6株能稳定分泌抗B型金黄色葡萄球菌肠毒素单克隆抗体的杂交瘤细胞株并进行了鉴定。结果6株单克隆抗体除两株属于Ig G2A外，其余均属于IGG1亚类，其培养上清和腹水的滴度分别为1：256～1：512和1：12800～1：25600，6株单克隆抗体均不与SEA发生交叉反应，仅有2株与SEC1有弱交叉反应。WESTERN-BLOT实验显示出一条特异性带，位于分子量28.5KDA处．证明了其特异性。结论成功获得抗B型金黄色葡萄球菌肠毒素单克隆抗体，为检测SEB打下了基础。     

Production and characterisation of monoclonal antibodies for species diagnosis of sarcosporidia

Monoclonal antibodies were raised against cystozoites of Sarcocystis muris and characterised. Twelve monoclonal antibodies reacted in the ELISA, Dot-ELISA and IFAT only with homologous antigen. The other twelve showed cross reactions of various degrees with cystozoites of S. gigantea, S. tenella, S. arieticanis, S. capracanis, S. miescheriana and S. suihominis. Proteins of S. arieticanis, S. tenella, S. gigantea, S. capracanis, S. muris and Toxoplasma gondii were compared by isoelectric focusing and showed pronounced differences.     

抗微囊藻毒素单克隆抗体胶体金标记物的制备及鉴定

目的制备抗微囊藻毒素(MC-LR)单克隆抗体胶体金标记物,并利用光谱分析来探讨结合物的合成机理。方法用杂交瘤技术生产并纯化的抗MC-LR的单克隆抗体,利用胶体金标记技术合成20nm粒径的胶体金与抗MC-LR单克隆抗体的结合物,通过光谱分析对偶联物中单克隆抗体的构象进行分析,ELISA法检测偶联物中抗体的免疫性。结果抗MC-LR单克隆抗体的效价达108,IC50抑制浓度为3ng/ml,为IgG2a亚型,分子量为1·5×105,与MC-LW、MC-LF无明显交叉反应。胶体金与抗体偶联物的颗粒圆整,光谱分析结果显示抗体蛋白的特征官能团没有发生明显变化,与胶体金粒子作用后结构略加紧密。ELISA结果显示偶联物仍然保持免疫原性。结论抗MC-LR单克隆抗体胶体金标记偶联物中抗体蛋白结构和抗原决定簇的位点没有改变,仍具有免疫原性,偶联物的稳定性良好。     

Immunochemical characterization of ovomucoid from Japanese quail egg white using monoclonal antibodies

The ovomucoid of Japanese quail egg white is known as a proteinase inhibitor or protein component responsible for egg allergy. In order to characterize the antigenic properties of ovomucoid in relation to its molecular structure, we have prepared two monoclonal antibodies, E9 (IgG1) and E11 (IgG1), recognizing distinct epitopes from each other. These monoclonal antibodies bound to the SDS/mercaptoethanol-treated ovomucoid, but not to the reductively carboxymethylated and pyridylethylated ovomucoid. By immunoblotting analysis of the peptic digests of ovomucoid, it was shown that E9 bound to the fragment consisting of two domains, I and II, of the ovomucoid, and that E11 reacted with the fragment containing domain III. These results indicate that the antigenic activity depends on the conformational structure of domains tightly folded by disulfide linkages. Ovomucoids from hen and duck were also recognized by both the antibodies, although having less affinity compared to the one from Japanese quail. These antibodies proved to be effective in establishing an enzyme-linked immunosorbent assay system for quantitative analysis of quail ovomucoid.     

鳙鱼变应原小清蛋白单克隆抗体制备及鉴定

目的 制备、纯化及鉴定抗鳙鱼小清蛋白单克隆抗体。方法 以重组鱼主要过敏原小清蛋白(parvalbu-min)为抗原免疫BALB/c小鼠,融合免疫鼠脾细胞和小鼠骨髓瘤NS-1,半固体培养基法结合有限稀释法筛选稳定分泌抗体的杂交瘤细胞株,杂交瘤细胞株诱生小鼠腹水,采用蛋白A亲和层析法获得纯化抗体,间接ELISA方法和w estern blot鉴定抗体效价和特异性以及与其他过敏源的交叉反应性;采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型。结果 共获得抗parvalbumin细胞株7株,1G1,1B5,1A5,2B9,1H4,1B7,1G3。1G1、1B7、1A5、1H4和1G3效价均高于1:400 000,P/N>2.1;1B5效价高于1:200 000,2B9效价较低;Western blot检测表明所有抗体均能识别parvalbumin;抗体亚类为IgG1型,1G1,1A5,2B9,1H4,1B7特异性结合parvalbumin,与其他种类过敏原无交叉反应,1G3和1B5与虾和大豆过敏原有交叉反应性,P/N>2.1。结论 获得高效价抗体5株,发现parvalbumin与虾、大豆过敏原存在交叉反应性,为建立parvalbumin检测体系提供依据。     

出血性大肠杆菌单克隆抗体的制备与鉴定

以肠出血性大肠埃希氏菌 (EnterohaemorrhagicE .coli,EHEC)O15 7∶H7免疫Balb c小鼠 ,取其脾细胞与小鼠骨髓瘤细胞Sp2 0融合、筛选 ,经 3次亚克隆建立了一个稳定分泌抗大肠杆菌O15 7单克隆抗体的杂交瘤细胞株 ,命名为 3A5。分泌抗体属IgM亚型 ,腹水的抗体效价达 1∶1× 10 6 。以辛酸 饱和硫酸铵法纯化后抗体的工作浓度为 1∶2× 10 4 ,检出限为 10 5～ 10 6 cfu ml。该抗体对大肠杆菌O15 7有较高特异性 ,同时抗出血性大肠杆菌O113∶H2 1抗原     

The pharmacokinetics of monoclonal antibodies

Monoclonal antibodies (MOABs) are, due to their specificity, increasingly being deployed for therapeutic purposes. MOABs are derived from immunoglobulins and are fully or partially of murine or human origin. They are administered parenterally: mostly intravenously, but subcutaneous or intramuscular administration is also possible, in which case absorption probably occurs through the lymphatic system. The distribution of MOABs from the bloodstream into the tissues is slow and is hampered by the high molecular size of the MOABs, which is a lesser problem for fragments of antibodies (Fab fragments). MOABs are metabolised to peptides and amino acids. This process takes place in many tissues of the body, but probably predominantly in epithelial cells. As a consequence of the saturable binding of the MOAB to its target, a dose-dependent (non-linear) elimination is often observed. Immune reactions can accelerate the elimination of antibodies, partially depending on the degree ofhumanisation of the antibody. Antibodies and endogenous immunoglobulins are protected from elimination by binding to protective receptors (neonatal Fc-receptor; FcRn), which explains their long half-lives (up to 4 weeks). Metabolic pharmacokinetic interactions with other drugs have not been reported and are not expected. It is expected that in the years to come, new MOABs directed towards new targets will appear on the market, as well as existing antibodies with improved pharmacokinetic properties.     

First characterization in Italy of clinical isolates of mutans streptococci by using specific monoclonal antibodies

The aim of this investigation was to gain further insight into the prevalence of different serotypes of mutans streptococci in the Italian population by using specific monoclonal antibodies in an enzyme immunoassay. Isolates from dental plaque samples, collected from an adult population living in Pisa (Italy), were identified as mutans streptococci on the basis of their morphological and biochemical properties, and were then serotyped. The results show that 77.5% of the strains isolated belonged to serotype c or f (i.e., S. mutans), 15.9% were serotype e (i.e., S. mutans) and only two strains (1.4%) belonged to serotype g (i.e., S. sobrinus). These data are partially in agreement with other studies in Europe and in the U.S.A. The distribution pattern of the various serotypes turned out to be substantially similar among the different groups of patients, subdivided on the basis of their caries status, indicating that none of the serotypes was particularly associated with dental caries.     

Production of mouse monoclonal antibodies against Helicobacter pylori Catalase and mapping the antigenic epitope by phage display library

The Catalase of Helicobacter pylori (H. pylori) helps bacteria to protect themselves from oxygen toxicity and damage and have been identified an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against Catalase and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, MAbs were produced by the hybridoma technique using recombinant Catalase--GST as the immunogen and were immunoscreened against phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 34 phage clones were randomly selected and their specificity to mAb was verified by sandwich and competitive inhibition ELISA. Fifteen phage clones were sequenced and their amino acids were deduced. One mimotope (SVSLPYANLATH) showed good match with Catalase protein at 394-405aa and the serum of mice induced by the phage clone clearly recognized Catalase protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Catalase would provide an alternative approach for the development of a vaccine for H. pylori.     

Characterization of monoclonal antibodies to Toxoplasma gondii

Proteins of Toxoplasma lysate were separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. The range of protein bands was 6-100 kD. Monoclonal antibodies 1B2, 5B10, 4F6 reacted with antigens of 21 kD, 35 kD and 66 kD. Monoclonal antibodies reacted with major antigens of the RH-tachyzoiten surface and HanR-cysts.     

Cytotoxicity of monoclonal antibodies to Trypanosoma brucei

Monoclonal antibodies (McAbs) were raised against Metacyclic Variable Antigen Types (M-VATs) of the AnTAR 1 and ETAR 1 serodemes of Trypanosoma brucei. Two dominant M-VATs, one from each serodeme, were labelled by two of the McAbs using the indirect immunofluorescence technique. These McAbs were of the IgM class, and labelled exposed epitopes on living trypanosomes. They showed lytic activity in vitro towards their respective homologous VAT trypanosomes, both in the presence and absence of complement. In vivo, the McAbs promoted lysis and clearance of trypanosomes from the bloodstream of infected mice. Prevention of reinfection with trypanosomes expressing the same VAT was conferred by the McAbs.     

抗苗勒管激素的单克隆抗体制备与鉴定

目的表达高纯度的抗苗勒管激素蛋白,以该蛋白作为抗原制备单克隆抗体,并鉴定抗体亚型,为进一步开发优质的抗苗勒管激素诊断试剂盒做技术储备。方法在表达载体pET-28a(+)质粒中接入经密码子优化的抗苗勒管激素基因(AMH)片段,转化于BL21细菌中,异丙基硫代-β-D-半乳糖苷(IPTG)诱导重组蛋白大量表达,对上清中可溶性蛋白进行纯化。使用SDS-PAGE分析纯化蛋白的纯度。经纯化的抗原蛋白3次皮下免疫小鼠,挑选产生高效价的抗体的小鼠,取其脾细胞,制备悬液。经细胞杂交技术与骨髓瘤细胞进行融合,筛选能稳定持久分泌抗AMH抗体的杂交瘤细胞株,并制备腹水抗体。通过亲和层析技术、BCA蛋白检测方法等测定手段,纯化抗体、检测抗体浓度。结果筛选到2株能持久分泌抗AMH蛋白单克隆抗体的杂交瘤细胞株,分别为克隆细胞株3F6和4E7,分泌抗体均为IgG1亚型。结论筛选出稳定分泌高纯度的抗苗勒管激素抗体的2株杂交瘤细胞,并制备单克隆抗体。下一步将其应用于AMH检测试剂盒的开发。     

Determination of Legionella antigens using monoclonal antibodies

The detection of antigens is the most important tool for rapid diagnosis of Legionellosis. 34 Legionella (L.) spp. with 51 serogroups have been identified from several sources. According to the antigenic diversity, it is necessary to select monoclonal antibodies (mab) adequate to diagnostic purposes. Mab with specificity to genus, species and serogroups were discussed. L. pneumophila accounts for 70 to 80% of all cases of Legionelloses. In this study self-made mab to L. pneumophila are presented that demonstrate these bacteria in clinical materials from respiratory tract using immunofluorescent tests and by detection of soluble antigens in pleura fluids and urine specimens using enzyme immunoassay.