牙龈卟啉单胞菌及其细胞外膜泡多克隆抗体的比较研究

分别制备牙龈卟啉单胞菌４７Ａ－ １ 及其膜泡的多克隆抗体,并用间接免疫荧光法对６７ 个临床标本进行检测。结果经统计学检验,两种抗体显著相关,Ｐ＜ ０．０１ ,符合率为８２．１ ％ ;膜泡抗体的检出率高于菌体抗体,Ｐ＜０．０５ ;膜泡抗体相对于菌体抗体的敏感性为９３．５ ％ 。膜泡抗体与牙龈卟啉单胞菌以外的其他口腔细菌无交叉反应,说明膜泡抗体的特异性较强。     

噬菌体单链抗体文库的构建及筛选

噬菌体抗体是继多克隆抗体、单克隆抗体之后兴起的第3代基因工程抗体.噬菌体抗体库技术是抗体基因文库技术和噬菌体表面展示技术相结合形成的一项新技术与方法,在生物科学领域极具潜力.现主要就近年来该技术在抗体基因扩增、抗体库的构建、筛选方法等方面的进展进行综述.     

Studies on the Neutralizing Antibody to Herpes Simplex Virus

The type-specific antibody responses in rabbits immunized with both types of herpes simplex virus (HSV), measured by complement-requiring neutralizing (CRN) and slow-reacting CRN (s-CRN) antibody assays were compared. Titers of type-specific antibody measured by s-CRN antibody assay were always markedly higher than those measured by CRN antibody assay. The s-CRN antibody assay was so sensitive that even an undetectable level of CRN antibody could be readily detected by this method. The detection of type-specific antibody by s-CRN antibody assay may be useful when attempting to analyze human HSV infection.     

重组抗体药物研究进展及应用

重组抗体药物的发展经历了鼠源单克隆抗体（McAb）、人 鼠嵌合抗体、人源化抗体和全人抗体等阶段,目前初步应用于抗肿瘤、抗自身免疫病、抗感染等领域。保持和提高抗体的亲和力、降低抗体的免疫原性是抗体药物基因工程改造的两大原则。在嵌合抗体成功的基础上,通过CDR移植、表面修饰、抗体库以及转基因鼠技术,逐步提高人源化程度至100%。然而,实验室水平的研究结果与实际应用仍然存在一定差距。就重组抗体药物的基本概况、现存的问题与可能的解决办法以及在肿瘤、病毒性疾病和阿尔茨海默病治疗上的应用情况等进行了综述。     

Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.     

抗体信息数据库

抗体是介导体液免疫的重要效应分子。近年来,随着治疗性抗体药物不断上市及其临床应用范围不断拓宽,治疗性抗体已成为生物制药的产业支柱。另一方面,随着抗体相关的基础研究日益深入和抗体序列、结构、功能表位等相关数据大量涌现,作为抗体数据管理、搜索与利用的重要工具,抗体资源库也层出不穷并得到长足发展,在相关的研究、开发、生产与销售中发挥日益重要的作用。本文对包括Kabat、IMGT、abYsis等在内抗体信息数据库进行介绍。     

The establishment of human anti-TROP2 antibody IgG and its inhibition function on pancreatic cancer cell proliferation

On the basis of anti-TROP2 Fab antibody, this study seek to construct a eukaryotic expression system of human anti-TROP2 antibody IgG, and to analyse the inhibition function of human anti-TROP2 antibody IgG in the cell proliferation of pancreatic cancer. The heavy and light chain genes of anti-TROP2 antibody were amplified respectively to establish the recombinant expression vector of human anti-TROP2 antibody IgG, named pWS-anti-TROP2. The human anti-TROP2 antibody IgG was obtained through transfecting the plasmids into the CHO dhfr^- cell line, selecting the monoclonal cell strains with high amounts of antibody expression by MTX screening and applying Protein G affinity in purification. The identification and immunologic activity of human anti-TROP2 antibody IgG were researched by Western Blot,SDS-PAGE, ELISA, immunofluorescence assay and flow cytometry method (FCM). MTT assay was conducted to analyse the inhibition effect of human anti-TROP2 antibody IgG on BxPC3 cell proliferation. The human anti-TROP2 antibody IgG eukaryotic expression system was established successfully to express human anti-TROP2 antibody IgG, in which the molecular weight of heavy chain and light chain were consistent with expectation, and it could specifically combine with TROP2 protein, the antibody titer reached 1:6,400. The MTT assay results indicated that human anti-TROP2 antibody IgG had a significant effect on inhibiting the proliferation of BxPC3 cell, and the inhibition function can be gradually increased with improved antibody dose and prolonged time. In the study, the human anti-TROP2 antibody IgG eukaryotic expression system was constructed successfully, the antibody could specifically bind to TROP2 protein on the surface of pancreatic cancer cells, and it is shown to have a significant inhibitory action in pancreatic cancer cell proliferation.     

噬菌体抗体库研究进展

治疗性抗体的发展经历了异源抗体、人源化抗体和人源性抗体几个阶段。目前,人源性抗体是治疗性抗体发展的主要方向,而噬菌体抗体库技术的出现为人源性抗体的制备提供了良好的技术平台,并且逐渐成为目前获得人源性抗体的主要手段之一。噬菌体抗体库技术是20世纪90年代初期抗体工程领域的一项重大进展,它是噬菌体展示和抗体库2种技术的集成,目前广泛应用于生物医学领域。本文对该技术的原理、类型、特点及研究进展进行了综述。     

离子交换色谱对抗VEGFR2单抗的电荷异质性分析

目的采用离子交换高效液相色谱(CEX-HPLC)分析抗VEGFR2(抗血管内皮生长因子受体2)单抗制品的电荷异质性。方法应用CEX-HPLC技术,对VEGFR2单抗进行电荷异质性分析,并结合羧肽酶B(CpB)和N-糖酰胺酶F(EndoF2)酶切,初步研究其电荷异质性的成因。结果用CEX-HPLC分析CpB酶切前后单抗,证明其碱性变异体主要由C末端赖氨酸不均一性引起;分析EndoF2酶切前后处理的单抗,证明其酸性变异体部分由N-糖末端上的唾液酸修饰所引起。通过2种酶的顺序酶切可相对准确地分析含C-末端赖氨酸以及含唾液酸单抗的比例。结论采用CEX-HPLC可较好地分析单抗的电荷异质性;并结合合适的酶切处理,可判断单抗主要电荷异质性的来源,为保证单抗制品生产工艺的稳定性及质量控制提供了有效手段。     

Production and characterization of a CD25-specific scFv-Fc antibody secreted from Pichia pastoris

Antibodies against CD25 would be novel tools for the diagnosis and treatment of adult T cell leukemia lymphoma (ATLL) and many other immune disorders. In our previous work, we successfully produced the single-chain fragment of a variable antibody against CD25, the Dmab(scFv) antibody, using Pichia pastoris. Here, we describe a novel form of an antibody against CD25, the Dmab(scFv)-Fc antibody, also produced by P. pastoris. To construct the Dmab(scFv)-Fc antibody, the Dmab(scFv) antibody was genetically fused to the Fc fragment of a human IgG1 antibody. A fusion gene encoding Dmab(scFv)-Fc antibody was cloned into the pPIC9K plasmid and expressed at high levels, 60–70 mg/l, by P. pastoris under optimized conditions. The Dmab(scFv)-Fc antibody was similar to the Dmab(scFv) antibody in its binding specificity but different in its molecular form and Fc-mediated effector functions. The Dmab(scFv)-Fc antibody and the Dmab(scFv) antibody both bound to CD25-positive MJ cells but not to CD25-negative K562 cells. The Dmab(scFv)-Fc antibody existed as a dimer whereas the Dmab(scFv) antibody was a monomer because it lacks the Fc fragment. The Dmab(scFv)-Fc antibody enhanced the antibody-dependent cellular cytotoxicity of CD25-positive cancer cells, whereas the Dmab(scFv) antibody was inactive in the antibody-dependent cellular cytotoxicity assays. In addition, compared to the Dmab(scFv) antibody, the Dmab(scFv)-Fc antibody showed stronger immunosuppressive activity in the Con A-stimulated lymphocyte proliferation system and in the mixed lymphocyte reaction system. These results demonstrate that the Dmab(scFv)-Fc antibody produced in P. pastoris is functional, and therefore it might be developed as a novel diagnostic and therapeutic tool for ATLL and other immune disorders.     

Naturally occurring subclinical Corynebacterium kutscheri infection in laboratory rats: strain and age related antibody response

Naturally occurring subclinical Corynebacterium kutscheri infection was analyzed by antibody response related to the strain of rats. Wistar-Lewis, Wistar and Spraque-Dawley rats were high responders in seroconversion rates and antibody titers, while Brown Norway and Fischer rats were low responders. The antibody response was related to age also. Some young rats had maternal antibody to C. kutscheri, but antibody disappeared before 8 weeks of age. Rats were antibody-negative for several months thereafter and became antibody-positive after 6 months of age. The antibody response was highest at 8 to 9 months of age in subclinical C. kutscheri infection. This antibody response was very late, compared to the antibody response to Sendai virus and Mycoplasma infections.     

Membrane lipid composition modulates the binding specificity of a monoclonal antibody against liposomes

A hybridoma secreting a monoclonal IgM 'anti-liposome' antibody was produced after injecting a mouse with liposomes containing dipalmitoylphosphatidylcholine, cholesterol, dicetyl phosphate, and lipid A. The antibody was selected by assaying for complement-dependent damage to liposomes lacking lipid A. The monoclonal antibody reacted best with liposomes containing the original immunizing mixture of lipids. Deletion of individual lipid constituents from liposomes diminished the ability of the liposomes to bind (adsorb) the antibody. Binding of the antibody was enhanced by including lipid A or galactosylceramide in the lipid bilayer, or by substituting egg phosphatidylcholine for dimyristoyl- (or dipalmitoyl-) phosphatidylcholine. Sphingomyelin could be substituted for dimyristoylphosphatidylcholine without altering the adsorption of antibody. Although the monoclonal anti-liposome antibody was completely inhibited by phosphocholine, it was probably not a conventional anti-phosphocholine antibody. The antibody apparently had a partial specificity for phosphate, and was inhibited by glycerophosphocholine, glycerophosphate, sodium phosphate, sodium sulfate, and inositol hexaphosphate, but not by choline or inositol.     

Efficiency and sensitivity of indirect immunoperoxidase methods

The peroxidase-antiperoxidase (PAP) complex method has repeatedly been claimed to be more sensitive and antibody efficient than the indirect peroxidase labeled antibody method. However, most studies comparing these methods used tissue sections as the test material. However, test systems with known amounts of antigen will allow more reliable comparison of these methods and quantitative evaluation of method sensitivity. We therefore compared the antibody efficiency and sensitivity of these methods for the detection of human chorionic gonadotropin in an enzyme linked immunosorbent assay (ELISA), an antigen spot test (AST) and tissue sections of choriocarcinoma. In the PAP technique rabbit PAP and goat anti-rabbit antibody were applied. The same antibody was peroxidase-labeled with the periodate technique and used in the labeled antibody method. In the ELISA the PAP method resulted in slightly higher antibody efficiency than the labeled antibody method. At low primary antibody dilutions the intensity of the reaction decreased with the PAP method but remained high with the labeled antibody method, in the ELISA as well as on tissue sections. In the AST the labeled antibody method and the PAP method appeared to be equally sensitive.     

Specificity controls for immunocytochemical methods.

Immunocytochemistry is used for antibody localization of proteins in cells and tissues. The specificity of the results depends on two independent criteria: the specificity of the antibody and of the method used. The antibody specificity is best determined by immunoblot and or immunoprecipitation. Absorption of the antibody with a protein does not determine that the antibody would have bound to the same protein in the tissue, and therefore is not a good control for antibody specificity. The specificity of the method is best determined by both a negative control, replacing the primary antibody with serum, and a positive control, using the antibody with cells known to contain the protein. With the increasing use of immunocytochemistry, it is important to be aware of the appropriate controls needed to show specificity of the labeling. (J Histochem Cytochem 48:163-165, 2000)     

Regulation of the immune response by antibody. II. The effects of different classes of passive guinea pig antibody on humoral and cell-mediated immunity in rats

The ability of different classes of passively administered guinea pig antibody (γ1, γ2, and IgM) to regulate humoral and cell-mediated immunity to flagellin, polymerized flagellin (POL), and sheep red blood cells (SRBC) was investigated in rats. It was found that at high concentrations, all classes of antibody suppressed the primary antibody responses and usually enhanced the delayed-type hypersensitivity induced by the three antigens. With flagellin and SRBC, the different classes of passive antibody varied in their suppressing and enhancing properties, being in the order: γ2 > γ1 = IgM. At low concentrations, γ1 and IgM enhanced the primary antibody response and suppressed the delayed hypersensitivity induced by flagellin. Such an effect was not observed with either POL or SRBC. Priming for a secondary antibody response was less readily suppressed by all classes of passive antibody. The removal of macrophage cytophilic antibody from γ2 converted this antibody to a preparation (γ2 absorbed) which had effects on humoral and cell-mediated immunity approaching that of γ1 antibody.     

Passive immunity to bovine rotavirus infection associated with transfer of serum antibody into the intestinal lumen.

The effect of circulating passive antibody on immunity to bovine rotavirus infections in neonatal calves was investigated. In the first experiment, rotavirus antibody titers in the small intestinal lumina of 5- and 10-day-old calves with a wide range of serum rotavirus antibody titers were determined. Neutralizing antibody was present in the small intestinal lumina in titers that correlated with the calves' serum titers (r = +0.84, P less than 0.01). Immunoglobulin G1 was the predominant isotype of intestinal luminal rotavirus antibody. Calves not fed colostrum during the absorptive period lacked rotavirus antibody in circulation and in the intestinal lumen at 7 days of age, even when they were fed large volumes of colostrum with a high rotavirus antibody titer at 48 h after birth. Therefore, rotavirus antibody is not retained in the intestinal lumen for 5 days following a colostrum meal, and the luminal antibody in the 5- and 10-day-old seropositive calves were probably derived from circulating antibody. In a second experiment, calves were passively immunized by subcutaneous injection of colostral whey with a high immunoglobulin G1 rotavirus antibody titer and challenged with virulent bovine rotavirus 48 h later. The passively immunized calves were protected from rotavirus infection and diarrhea compared with calves with comparable serum immunoglobulin concentrations but with lower serum rotavirus with lower serum rotavirus antibody titers. The results of these experiments indicate that circulating immunoglobulin G1 antibody appears in the gastrointestinal tract of neonatal calves and that circulating rotavirus antibody can prevent infection and diarrhea after rotavirus challenge.     

Leucocyte myosin and its location in the cell.

The intracellular location of the binding site of antibody against purified myosin prepared from equine leucocytes was investigated in neutrophils and lymphocytes by electron microscopy using peroxidase-labelled antibody method. The myosin extracted from equine leucocytes could bind skeletal muscle F-actin and the formed complex showed the biophysical and biochemical properties and electron microscopic appearance of actomyosin. On immunodiffusion, the leucocyte myosin formed a single precipitin line with its antibody prepared in rabbits. The antibody also formed single precipitin lines with myosins from lymphocytes and thrombocytes, fusing with each other. The antibody against the leucocyte myosin did not react with myosins from skeletal or arterial smooth muscle. The specificity of the antibody was further established by determination of K+-EDTA-activated ATPase activity remained in the supernate of antigen-antibody mixture. Under electron microscope, the intracellular immunoreactive products of peroxidase labelled antibody were found in cytoplasm of neutrophils and lymphocytes incubated with antibody against leucocyte myosin, but not in neutrophils or lymphocytes treated with IgG from normal rabbits.     

抗CD3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定

利用基因工程方法将鼠源性抗CD3抗体HIT3a的可变区和人源抗体(IgG)的完整的恒定区连接起来,构建全抗型抗CD3嵌合抗体,该型抗体具有较低的免疫源性可作为免疫抑制剂应用于器官移植,减少受体产生免疫排斥,提高移植器官的存活率。利用PCR方法从抗CD3 ScFv重组噬菌体表达载体pCANTAB 5E上扩增抗CD₃抗体的轻链和重链可变区,将轻链和重链可变区组装到含有人抗体（IgG）恒定区的表达载体中,构建抗CD3嵌合抗体IgG的轻链和重链表达载体PKN100和PG1D105,并用脂质体法共转染CHO细胞。结果证明,抗CD₃嵌合抗体的V_L和V_H与HIT3a抗体的V_L和V_H完全相符,ELISA和Western blot检测结果证实转染细胞的培养上清中含有抗CD₃嵌合抗体IgG的表达,表达产物能与Jurkat细胞结合,并能竞争性抑制HIT3a抗体和Jurkat细胞结合活性,³H-TdR掺入实验表明, 抗CD3嵌合抗体与亲代抗体HIT3a一样,具有促进外周血单核细胞增殖的作用。我室构建的全抗型抗CD3嵌合抗体分子表达载体可在CHO细胞中稳定表达,表达产物有较好生物活性,具有潜在的临床应用价值。     

用间接免疫荧光法检测流行性出血热病人尿中特异性抗体的研究

用间接免疫荧光法检测110例不同病程、病期及病型的流行性出血热病人尿中及血清中特异性抗体。尿中IgM型抗体阳性率为62.7％。尿中IgG型抗体阳性率91.8％与血清IgG型者90.9％相似,而总阳性率(IgG或IgM有一项以上阳性者的总检出率)99.1％则高于血清IgG者。20例其它疾病及10例正常人尿抗体均为阴性。结果表明尿抗体检查法是特异且可靠的,它比血清学方法简便、灵敏、为临床诊断可早期快速得出结果,不用采血有利于病人。IgM型抗体阳性率受病程、病期、病型及尿蛋白量的影响较明显。     

Improved radioimmunodetection of tumours using liposome-entrapped antibody

The discrimination of radioimmunodetection of tumours is reduced by the presence of circulating radiolabelled antibody (primary antibody). We have prepared liposomes containing an antibody to the primary antibody (secondary antibody), with the intention of complexing and delivering to the liver primary antibody which is not associated with the tumour. In mice bearing xenografts of human tumours which secrete the marker carcinoembryonic antigen (CEA), liposomally entrapped secondary antibody was able to reduce the blood levels of 125I-labelled anti-CEA within 2 h, without reducing the amount of anti-CEA bound to the tumour. We therefore suggest that the use of liposomally entrapped secondary antibody would improve the diagnostic potential of radioimmunodetection of tumours and their metastases.