抗猪促卵泡激素小鼠源单克隆抗体的研究

用猪促卵泡激素(PESH)作为抗原免疫BALB/C小鼠。通过将免疫小鼠的脾细胞和同系小鼠骨髓瘤细胞(SP2/O—Ag14)进行融合,建立了九株能稳定分泌抗PFSH单克隆抗体的杂交瘤细胞。其中三株杂交瘤细胞分泌的抗体被证实是对PFSH特异结合的,它们和另一种非常相近的垂体激素——猪促黄体激素(PLH)不结合。用有限稀释法对全部九株杂交瘤进行了三次亚克隆。有几种单克隆抗体被羟基磷灰石柱层析一步法从小鼠腹水所纯化。以聚丙烯酰胺凝胶电泳法检测经纯化单克隆抗体制品的纯度。     

柑桔速衰病毒株系特异性单克隆抗体的制备及其免疫学特性

以感染枸橼的柑桔速衰病毒(Citrustristeza virus,CTV)的部分纯化制备物和浓缩的感染组织粗浸出液免疫BALB/C小白鼠后获得的脾脏细胞,与鼠骨髓瘤细胞株P3-X63-Ag8.653融合制备了杂交瘤细胞.免疫小鼠的CTV株系为T514,杂交瘤细胞以有限稀释法获得纯克隆,并以健康枸橼和CTV不同株系感染枸橼的组织浸出液筛选各克隆,并确认各克隆与CTV各株系的免疫反应性.经以碱性磷酸酶标记的山羊抗鼠IgG进行酶联免疫吸附试验(EUSA),对各杂交瘤克隆的株系特殊性分析证明,从这些杂交瘤细胞中获得了能产生CTV株系特异性单克隆抗体的杂交瘤细胞株.     

抗人癌胚抗原单克隆抗体的研制及其在免疫组化中的应用

以高纯CEA为抗原,免疫BALB／c小鼠．取其脾细胞和小鼠SP2／0-Ag14骨髓瘤细胞融合．经2次亚克隆。建立3株稳定分泌抗CEA特异性单克隆抗体的杂交瘤细胞株。应用经纯化的该单克隆抗体进行了结肠癌组织切片的免疫组织化学实验．结果显示全部3种抗CEA单克隆抗体对结肠癌亚细胞组分均呈现特异性结合。表明已筛选出适于制作癌胚抗原临床诊断试剂盒所需的特异性单克隆抗体。     

Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice

Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.     

人白血病抑制因子（hLIF）单克隆抗体的制备与鉴定

 为了获得抗人白血病抑制因子（hLIF）单克隆抗体,以重组hLIF蛋白为抗原,免疫Balb/c小鼠,采用传统的小细胞融合、无限稀释法制备了10株杂交瘤的腹水hLIF抗体,并对腹水进行了纯化.腹水粗品抗体经rProteinA一步亲和层析法纯化和鉴定后,抗体分子量都符合抗体的特性,抗体的纯度都达到了96.6%以上,抗体亲和常数在1.89×10-9~1.51×10-12mol/L,其中1B8a、5C1b、5G4a细胞株制备的抗体属于高亲和力抗体,可以对其的应用进行进一步的研究和探讨.     

抗甲胎蛋白单克隆抗体的研制及其在免疫组化中的应用

以高纯AFP为抗原,免疫BALB/c小鼠,取其脾细胞和小鼠SP2/0 Ag14骨髓瘤细胞融 合,经2次亚克隆,建立5株稳定分泌抗AFP特异性单克隆抗体的杂交瘤细胞株。应用经纯化 的该单克隆抗体进行了原发性肝癌组织切片的免疫组织化学实验,结果显示全部5种抗AFP单 克隆抗体对肝癌亚细胞组分均呈现特异性结合。表明已筛选出适于制作甲胎蛋白临床诊断试 剂盒所需的特异性单克隆抗体。     

抗棉铃虫组织蛋白酶B单克隆抗体的制备及鉴定

分别以棉铃虫组织表达的组织蛋白酶B和基因重组的大肠杆菌表达的组织蛋白酶B为抗原免疫BALB／c小鼠，选择血清抗体滴度高的免疫小鼠，取其脾细胞和Sp2／0骨髓瘤细胞进行细胞融合．经ELISA法筛选阳性克隆和有限稀释法克隆细胞，共获得分泌抗组织蛋白酶B的单克隆抗体杂交瘤细胞3株，分别命名为9D5，100E2和100H6．以ELISA法和Western blotting法对3株杂交瘤细胞株分泌的单克隆抗体进行了特异性鉴定，其中9D5和100E2能够同时识别两种不同来源的组织蛋白酶B，而100H6只能识别基因重组的大肠杆菌表达的组织蛋白酶B．     

Phage antibodies: filamentous phage displaying antibody variable domains

New ways of making antibodies have recently been demonstrated using gene technology. Immunoglobulin variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors. Soluble antibody fragments secreted from bacteria are then screened for binding activities. Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage. Phage carrying V genes that encode binding activities could then be selected directly with antigen. Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.     

兔抗人多发性骨髓瘤细胞多克隆抗体的制备与鉴定

制备人多发性骨髓瘤全细胞兔多克隆抗体。用人多发性骨髓瘤细胞系ARH-77全细胞和福氏完全佐剂通过背部皮内多点注射首次免疫新西兰大白兔,第14 d用ARH-77全细胞和福氏不完全佐剂同样剂量加强免疫,第28 d和38 d时再次免疫。第45 d颈动脉采血80 mL,4℃静置过夜,收集血清。然后采用亲和层析系统纯化血清中的多克隆抗体。用酶联免疫吸附试验(ELISA)检测纯化多克隆抗体的效价;免疫印迹法和荧光免疫细胞化学检测纯化多克隆抗体的特异性。结果:ELISA检测多克隆抗体滴度为1∶20 000,免疫印迹检测结果显示该抗体具有较高特异性,荧光免疫细胞化学检测多克隆抗体能够有效的结合细胞表面抗原。实验获得了高效价的特异性的兔抗人骨髓瘤细胞多克隆抗体,为进一步研究多发性骨髓瘤诊断和治疗奠定了基础。     

T-cell hybridoma bearing heavy chain variable region determinants producing (T,G)-A--L-specific helper factor

Thymus-derived lymphocytes (T cells) exert their regulatory effect (help or suppression) on the antibody production by B cells either by direct cell to cell interaction or by soluble mediators or factors. The low frequency of specific T cells, the heterogeneity of their responses and their relatively short life span have hampered the molecular characterization of the antigen recognition unit of T cells, and its structure is largely unknown. The lymphocyte hybridization technique, which has been found very useful for the production of B-cell hybridomas secreting specific monoclonal antibodies, has also been used for the generation of homogeneous and stable T-cell hybridomas with unlimited growth potential. So far the only specific effector function demonstrated in the established T hybridomas is the property to generate a factor(s) which suppresses antibody responses. We now describe the establishment of hybrid lines which exhibit characteristic T-cell markers. One of the hybridomas (denoted R-9) releases into the culture supernatant factor(s) with helper activity specific to the synthetic polypeptide (T,G)-A--L and bears surface determinants of the immunoglobulin heavy chain variable region (VH). Such hybrid cell lines are of great value for studies on the nature of the T-cell receptor.     

抗KDR-Fc杂交瘤细胞系的建立

用杂交瘤技术建立抗KDRFc的杂交瘤细胞系,以KDRFc抗原免疫雌性BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,经ELISA间接法检测和有限稀释法克隆培养,再经稳定性检测、效价检测,筛选出分泌抗KDRFc单克隆抗体的杂交瘤细胞.得到了10株能持续分泌抗KDRFc的单克隆抗体的细胞株,其中3株分泌的单抗具有较高的生物活性、特异性和稳定性.     

The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3

CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3] is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,000 (Mr 50-58 K) which appears early in thymocyte ontogeny and is present on all mature T cells. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes, cytotoxic effector function and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits rosetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated antigen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.     

Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein

Exocytotic secretion requires the interaction and fusion of secretory vesicles with the plasma membrane. This process could be mediated by specific recognition molecules acting as intracellular, membrane-bound receptors and ligands. One possible component of such a recognition site on the plasma membrane is a protein of relative molecular mass (Mr) 51,000 (51K) that has been isolated from bovine adrenal chromaffin cells. This protein binds strongly to chromaffin granules, the secretory vesicles of these cells. To determine the function of this membrane-anchored chromaffin granule-binding protein in exocytosis, we tested the effect of intracellularly injected antibodies on secretion. Here we show, by two independent techniques in two different cell types, that antibodies against this protein inhibit exocytosis. In rat pheochromocytoma cell cultures, monospecific antibodies, applied by erythrocyte ghost fusion, impair the release of 3H-noradrenaline. The same antibodies, introduced into individual chromaffin cells through a patch pipette, block exocytosis, as revealed by the measurement of membrane capacitance. These results demonstrate the functional involvement in exocytosis of a plasma membrane protein with high affinity for secretory vesicles.     

Monoclonal antibodies to the acetylcholine receptor by a normally functioning auto-anti-idiotypic mechanism

Recently we described a procedure for preparing antibodies to the acetylcholine receptor (AChR) based on immunoglobulin idiotypes and on the hypothesis that, regardless of functional differences, macromolecules of the same specificity will show structural homologies in their binding sites. Antibodies were prepared in rabbits to a structurally constrained agonist of AChR, trans-3,3'-bis[alpha-(trimethylammonio)methyl]azobenzene bromide (BisQ). These antibodies mimicked the binding specificity of AChR in its activated state--agonists were bound with affinities that were in accord with their biological activities and antagonists were bound poorly. Rabbits were then immunized with a specifically purified preparation of anti-BisQ to elicit a population of antibodies specific for the binding sites of anti-BisQ. A portion of the anti-idiotypic antibodies produced in the second set of rabbits cross-reacted with determinants on AChR preparations from Torpedo californica, Electrophorus electricus and rat muscle. Moreover, several of the rabbits showed signs of experimental myasthenia gravis, in which circulating AChR antibodies are typically found. To devise a more direct route to monoclonal anti-receptor antibodies we based our strategy on acceptance of the concept of the anti-idiotypic network theory of Jerne. According to this theory, injection of an antigen elicits, in addition to antibodies to the antigen, other populations that include anti-idiotypic antibodies directed at the combining sites of the antigen-specific antibodies. If the antigen-specific antibodies recognize a ligand of a receptor, then the anti-idiotypic antibodies should bind receptor. Thus, when a mouse is immunized with a bovine serum albumin conjugate of BisQ (BisQ-BSA), it should be possible to expand populations of spleen cells that secrete antibodies which bind anti-BisQ and AChR, in addition to populations specific for BisQ. Fusion of the spleen cells with an appropriate myeloma line should yield monoclonal anti-AChR antibodies. Here we report the success of this approach and its implications.     

HMG＿（14）单克隆抗体杂交瘤株的建立

以北京鸭红细胞提取的ＨＭＧｓ（ＨＭＧ＿１，ＨＭＧ＿（２ａ），ＨＭＧ＿（２ｂ），ＨＭＧ＿（１４），ＨＭＧ＿（１７））免疫Ｂａｌｂ／ｃ小鼠，取其脏压细胞与Ｓｐ２／０（小鼠骨髓瘤细胞）融合，经选择培养，阳性筛选，克隆化以及冻存复苏，获得了三株稳定分泌抗ＨＭＧ＿（１４）的杂交瘤细胞株（１Ｃ＿（１２），１Ｏ＿３和２Ｆ＿２），经ＷｅｓｔｅｒｎＢｌｏｔ鉴定，与其它几种ＨＭＧ无反应，抗体为ＩｇＧ＿１亚型。腹水ＨＭＧ＿（１４）单克隆抗体经ＳｅｐｈａｄａｘＣＬ－４Ｂ亲和柱得到了纯化。     

乙型脑炎病毒(JEV)单克隆抗体的制备及初步应用

目的制备乙型脑炎病毒(JEV)单克隆抗体,应用于猪源性生物制品中JEV的检测。方法提纯病毒抗原,免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞融合,经间接ELISA方法筛选和有限稀释法三次克隆获得4株杂交瘤细胞,并应用琼脂扩散、酶联免疫吸附试验和免疫荧光方法进行抗体鉴定和样品检测。结果获得4株稳定分泌JEV单克隆抗体的杂交瘤细胞株G10、F4、C10、A5,免疫荧光抗体实验证实是特异的抗JEV抗体,其免疫球蛋白亚类G10和F4为IgG1,A5为IgM,C10未鉴定出其亚类。G10和F4腹水效价为105以上,C10和A5腹水效价为102以上。结论成功制备了抗JEV单克隆抗体,建立了以单抗介导的间接ELISA和间接IFA检测方法,并应用于猪源性生物制品中JEV的检测。     

Interaction between CD4 and class II MHC molecules mediates cell adhesion

The CD4 glycoprotein is expressed on T-helper and cytotoxic lymphocytes which are restricted to class II major histocompatibility complex (MHC) antigens on target cells. Antibody inhibition studies imply that CD4 acts to increase the avidity of effector-target cell interactions. These observations have led to the speculation that CD4 binds to a monomorphic class II antigen determinant, thereby augmenting low affinity T-cell receptor-antigen interactions. However, no direct evidence has been presented indicating that CD4 and class II molecules interact. To address this issue, we have used a vector derived from simian virus 40 (SV40) to express a complementary DNA (cDNA) encoding the human CD4 glycoprotein. When CV1 cells expressing large amounts of the CD4 protein at the cell surface are incubated with human B cells bearing MHC-encoded class II molecules, they are bound tightly to the infected monolayer, whereas mutant B cells which lack class II molecules fail to bind. Furthermore, the binding reaction is specifically inhibited by anti-class II and anti-CD4 antibodies. Thus, the CD4 protein, even in the absence of T-cell receptor-antigen interactions, can interact directly with class II antigens to function as a cell surface adhesion molecule.     

Monoclonal antibodies identify a cell-surface antigen associated with an activated cellular oncogene

A variety of antigens have been identified on the surface of the malignant cell. However, identical antigens are often found on non-malignant cells of the same or different histological origin, or of a different stage of embryonic development. Many of these tumour-associated antigens appear to be only incidentally expressed on neoplastic cells. Clearly, it would be of great interest to identify cell-surface antigens whose expression is associated specifically with the transformed state and linked directly with the mechanisms responsible for transformation. The detection of activated cellular oncogenes in human and animal cancer cells by the technique of DNA transfection has allowed the isolation of genetic elements which are thought to have a critical role in malignancy. Here, in an effort to identify cell-surface antigens associated with the neoplastic process, we have generated hybridomas which secrete monoclonal antibodies that react specifically with cell-surface determinants found on NIH 3T3 cells transformed by transfection with a group of rat neuroblastoma oncogenes. These antibodies bind to and immunoprecipitate a phosphoprotein of relative molecular mass 185,000 (185 K) from a DNA donor rat neuroblastoma and 13 independent rat neuroblastoma DNA transfectants. There was no antibody reactivity with normal NIH 3T3 cells or with NIH 3T3 cells transformed by various other agents.     

尼罗罗非鱼IgM单抗的制备及IgM+B淋巴细胞吞噬能力的研究

采用免疫亲和层析方法纯化尼罗罗非鱼抗TNP特异性IgM，并以此为抗原免疫Babl/c小鼠进行单克隆抗体制备. 免疫的小鼠脾脏细胞与骨髓瘤细胞(SP2/0)融合成杂交瘤细胞，利用酶联免疫吸附法、蛋白质印迹法(Western blot)和流式细胞术筛选获得两株抗IgM重链的单克隆抗体，分别命名为3B3和9D12. 纯化后的单克隆抗体3B3和9D12在1 mg/mL浓度下其抗体效价分别为740,741和359,712 units/mL. 利用制备的单抗结合激光共聚焦显微镜检测发现，膜结合型IgM位于B细胞膜表面(IgM+ B细胞). 流式细胞术检测尼罗罗非鱼IgM+ B细胞的免疫组织分布，发现IgM+ B细胞分布存在组织差异性，其中在外周血(PBL)所占比例最大，约为37.6%，其次是脾脏(SPL)，占百分比33.7%，头肾(AK)占比例约为23.9%，而后肾(PK)占比例约为20%. IgM+ B细胞荧光微球的吞噬能力分析发现，IgM+ B细胞对0.5 m的荧光微球吞噬能力强于1 m. 另外，IgM+ B细胞的吞噬能力存在免疫组织差异性，其中对于0.5 m荧光微球的吞噬，PBL明显高于其他组织，而AK IgM+ B细胞对1 m荧光微球的吞噬能力最强. 以上结果表明，本研究成功制备了鼠抗尼罗罗非鱼IgM单抗工具，并利用其IgM单抗发现IgM+ B细胞具有吞噬能力且其吞噬能力存在组织差异性，表明IgM+ B细胞在先天性免疫中可能发挥重要作用.     

Making antibody fragments using phage display libraries

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.