Drastic change in idiotypic but not antigen-binding specificity of an antibody by a single amino-acid substitution

In proliferating B lymphocytes, somatic mutation of rearranged antibody variable (V)-region genes occurs at high frequency and may have a key role in the selection of these cells. It is of interest in this context to learn in which way single mutations can affect antigen binding and/or idiotypic specificity of an antibody. Previous investigations have analysed spontaneous mutants of myeloma and hybridoma cells in which the mutation affected the antigen-binding specificity of the antibody. Here we describe an antibody mutant that has fully retained antigen-binding specificity but has lost or drastically changed all V-region antigenic determinants (idiotopes) of the wild type as defined by monoclonal anti-idiotope antibodies. The mutant phenotype is generated by a glycine to arginine exchange in the middle of the diversity (D) element, at position 103 of the heavy chain.     

Three-dimensional structure of an antigen-antibody complex at 6 A resolution

Present understanding of the three-dimensional structure of antibody combining sites is based on X-ray diffraction studies of myeloma immunoglobulins. The structures of the antigen-binding fragment (Fab) complexes of two of these immunoglobulins with small ligands have also been determined. However, there is no crystallographic information concerning the interactions of an antibody with an antigen, nor do we know the precise structure of antigenic determinants on protein molecules. We now report the first structure determination of an antigen-antibody complex at 6 A resolution. The structure of the complex between hen egg-white lysozyme and the Fab of a monoclonal anti-lysozyme antibody (D1.3) shows that the combining site of antibodies is not merely a cleft delineated by the complementarity-determining regions of the variable regions of the light and heavy chains, but is a larger area extending beyond it. A correspondingly large area of the antigen makes close contacts with the antibody, in agreement with the notion of a 'topographical' rather than 'sequential' antigenic determinant. The structural basis of cross-reactivities of an antibody with heterologous antigens and the effect of a single amino acid substitution on antigenic specificity can thus be visualized in the structural model presented here.     

Evidence for a physical association of CD4 and the CD3:alpha:beta T-cell receptor

CD4 is a molecule expressed on the surface of T lymphocytes which recognize foreign protein antigens in the context of class II major histocompatibility complex (MHC) molecules. Recognition of antigen:class II MHC complexes by CD4+ T cells can be inhibited by anti-CD4 (ref. 3). Nevertheless, specific recognition of the antigen:Ia complex is clearly a function of the T-cell receptor, which is composed of CD3 and the variable polypeptides alpha and beta. Thus, it has been proposed that CD4 serves an accessory function in the interaction of CD4+ T cells and Ia-bearing antigen-presenting cells by binding to non-polymorphic portions of class II MHC molecules and stabilizing the cell interaction. Based on our observation that anti-CD4 could inhibit activation of a cloned line of CD4+ T cells by antibodies directed at a particular epitope on the variable region of the T-cell receptor, we have recently proposed that CD4 is actually part of the T-cell antigen recognition complex, physically associated with CD3:alpha:beta. But numerous studies showing that CD3 and CD4 are not stably associated on the T-cell surface would appear to contradict this model. Here we show that anti-T-cell-receptor antibodies can co-modulate expression of the T-cell receptor and CD4, and that the monovalent Fab fragment of such an anti-T-cell-receptor antibody can, in conjunction with bivalent anti-CD4 antibody, generate an activating signal for the T cell. These findings provide further evidence for a physical association of the T-cell receptor complex and CD4.     

Production of antibodies in transgenic plants

Complementary DNAs derived from a mouse hybridoma messenger RNA were used to transform tobacco leaf segments followed by regeneration of mature plants. Plants expressing single gamma or kappa immunoglobulin chains were crossed to yield progeny in which both chains were expressed simultaneously. A functional antibody accumulated to 1.3% of total leaf protein in plants expressing full-length cDNAs containing leader sequences. Specific binding of the antigen recognized by these antibodies was similar to the hybridoma-derived antibody. Transformants having gamma- or kappa-chain cDNAs without leader sequences gave poor expression of the proteins. The increased abundance of both gamma- and kappa-chains in transformants expressing assembled gamma-kappa complexes was not reflected in increased mRNA levels. The results demonstrate that production of immunoglobulins and assembly of functional antibodies occurs very efficiently in tobacco. Assembly of subunits by sexual cross might be a generally applicable method for expression of heterologous multimers in plants.     

Immunomodulation of experimental allergic encephalomyelitis by antibodies to the antigen-Ia complex.

Autoimmune diseases occur when T lymphocytes become activated on recognizing self antigen linked to the autologous class II molecule of the major histocompatibility complex (MHC). The resulting complex of antigen MHC T-cell receptor could be a target for treatment of autoimmune diseases. Studies in which each component is blocked separately might be limited by interference in non-relevant immune responses that either use the same set of T-cell-receptor V gene segments or are linked to the same MHC. We report here an attack by a specific antibody on the unique antigenic site formed by the binding of two components of the trimolecular complex, the autoantigen bound to the self MHC. We tested its effect in experimental allergic encephalomyelitis, an acute neurological autoimmune disease which is widely regarded as a model for autoimmune disorders and which is mediated by CD4+ T cells recognizing myelin basic protein (BP), or its peptides, in association with self Ia. We made monoclonal antibodies which bound only the complex of BP and I-As. These antibodies blocked the proliferative response in vitro to the encephalitogenic determinant of BP and reduced the response to intact BP, without affecting the response to a nonrelevant antigen-purified protein derivative of tuberculin presented on syngeneic macrophages. They also inhibited experimental allergic encephalomyelitis in H-2s mice. Hence, antibodies directed specifically to the autoantigen-Ia complex, may offer a highly selective and effective treatment in autoimmune diseases.     

The binding site for C1q on IgG

In humoral defence, pathogens are cleared by antibodies acting as adaptor molecules: they bind to antigen and trigger clearance mechanisms such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and complement lysis. The first step in the complement cascade is the binding of C1q to the antibody. There are six heads on C1q, connected by collagen-like stems to a central stalk, and the isolated heads bind to the Fc portion of antibody rather weakly, with an affinity of 100 microM (ref. 3). Binding of antibody to multiple epitopes on an antigenic surface, aggregates the antibody and this facilitates the binding of several C1q heads, leading to an enhanced affinity of about 10 nM (ref. 1). Within the Fc portion of the antibody, C1q binds to the CH2 domain. The interaction is sensitive to ionic strength, and appears to be highly conserved throughout evolution as C1q reacts with IgG from different species (for example see ref. 8). By systematically altering surface residues in the mouse IgG2b isotype, we have localized the binding site for C1q to three side chains, Glu 318, Lys 320 and Lys 322. These residues are relatively conserved in other antibody isotypes, and a peptide mimic of this sequence is able to inhibit complement lysis. We propose that this sequence motif forms a common core in the interactions of IgG and C1q.     

BALB／c小鼠选择与腹水产量和抗体效价间关系的研究

目的观察在鼠疫F1单克隆抗体制备过程中,BALB／c小鼠的选择和饲养与所获腹水量和抗体效价的关系,为大量制备该单克隆抗体提供实验室参考依据。方法根据小鼠周龄、性别和饲养条件对BALB／c小鼠进行分组,并采用动物体内诱生法制备腹水,间接ELISA用来检测腹水中F1单克隆抗体效价。结果12～16周龄组、雄性和加强营养组小鼠所获腹水产量和效价均高于其它周龄组和对照组。结论选择适当周龄的雄性小鼠、饲养过程中加强营养和管理可适当提高腹水产量和抗体的效价。     

RH株弓形虫抗原及高效价多克隆抗体的制备

通过制备和分析弓形虫抗原蛋白，并以此抗原制备高效价的兔抗弓形虫多克隆抗体，为弓形虫的免疫检测和诊断提供有效的方法和途径，同时也为基因工程产物的鉴定提供方便有效的检测手段．速殖子主要来源于感染后72～74h获得的腹水，通过常规方法制备和提纯弓形虫可溶性抗原，采用SDS-AGE方法分析抗原蛋白的分子量．以抗原和福氏佐剂制备成完全和不完全福氏佐剂通过脊柱两旁皮下多点免疫的途径免疫新西兰大白兔，制备兔抗弓形体多克隆抗体，间接ELISA法检测血清中抗体的效价．经过观察所获得的虫体阶段主要处在宿主细胞巨噬细胞内而尚未逸出阶段，通过对结果的分析，所制备的弓形体抗原的分子量主要集中在17～156ku之间，与文献报道有差异，兔抗弓形体抗体效价在60000以上．该研究结果将为弓形体的蛋白质研究，免疫诊断、预防及有效治疗奠定基础．     

Immunization to a syngeneic sarcoma by a monoclonal auto-anti-idiotypic antibody

Idiotypic networks regulate the immune response to a variety of antigens. Antibodies generated against other antibodies, called anti-idiotypic antibodies, can themselves mimic antigen and elicit a specific immune response. They have been shown to induce delayed-type hypersensitivity (DTH) to model antigens in the mouse. As anti-idiotypic antibodies are thought to be involved in the response to tumour-associated antigens we tested whether injection of monoclonal antibodies derived from mice hyperimmunized to a syngeneic, chemically induced sarcoma could mimic antigen and induce DTH to the sarcoma in naive mice. One of the monoclonal antibodies, 4.72, primed BALB/c mice for DTH to the sarcoma but not for DTH to another sarcoma or to sheep erythrocytes. Antibody 4.72 did not induce DTH in mice of immunoglobulin allotype congeneic strains nor did it bind to the sarcoma cells. As antibodies specific for this sarcoma have not been detected, we do not know whether idiotype on immunoglobulin molecules is recognized by antibody 4.72. However, as the response induced by antibody 4.72 was both antigen-specific and allotype-restricted, analogous to those induced by anti-idiotypic antibodies in other systems, we propose that antibody 4.72 is an anti-idiotypic antibody.     

基于距离浓度的K-均值聚类算法

提出的基于距离浓度的K-均值聚类算法把聚类的数据对象视为抗原,聚类中心看作是免疫系统中的抗体,聚类过程表示为免疫系统不断产生抗体,识别抗原,最后产生出可以捕获抗原的最佳抗体过程.定义了抗体浓度和亲和度,使得抗体之间的距离越大,其距离浓度越小,反之则浓度越大,从而提高了算法的搜索效率.设计了抗体的期望繁殖率计算方法和克隆变异方法.仿真结果表明:该算法不仅克服了传统的K-均值聚类算法易陷入局部极小值的缺点,而且避免了对初始化选值敏感性的问题,同时也有较快的收敛速度.     

A new strategy for the generation of catalytic antibodies

The high binding affinity and specificity of antibodies for a wide range of ligands has recently been exploited in the generation of catalysts for acyl-transfer reactions, carbon-carbon bond forming and carbon-carbon bond cleaving reactions. In addition, a number of strategies are emerging for the generation of catalytic antibodies including transition state stabilization, catalysis by approximation, and the introduction of catalytic groups or cofactors into antibody combining sites. An important goal in the design of catalytic antibodies is the development of general rules relating hapten structure to the corresponding catalytic groups in the antibody combining site. We report here that electrostatic interactions between a hapten and the complementary antibody can be exploited to generate catalytic amino-acid side chains in an antibody-combining site. The antibody-catalysed reaction, a beta-elimination reaction, exhibits saturation kinetics, substrate specificity, competitive inhibition by hapten, and specific inactivation by a reagent that modifies carboxylate residues.     

Functional and molecular organisation of an antigen-specific suppressor factor from a T-cell hybridoma

Thymus-dependent (T) lymphocytes have been shown to have antigen specificity. The antigen receptor on T lymphocytes, in contrast to that on B lymphocytes, does not appear to be of the conventional immunoglobulin (Ig) type. Studies on the antigen-specific factors derived from helper and suppressor T cells (Ts) demonstrated that they possess determinants with antigen binding affinity and products of genes in the H-2 complex (MHC). Furthermore, antibodies against the variable region of Ig heavy chains or idiotypes have been shown to react with T-cell antigen receptors as well as antigen-specific helper and suppressor T-cell factors (TsF). It is, therefore, conceivable that at least two gene products are involved in the structural entity of these receptors: one each coded for by genes in either. To establish the molecular nature of the recognition component of T cells we have used homogeneous TsF from a T-cell hybridoma with a specific function. We report here that the antigen binding and I-J coded molecules on TsF are independently synthesised in the cytoplasm, and are secreted as an associated form of the two molecules; this association is required for antigen-specific suppression of antibody response.     

基于表面重塑方法的抗体HAb18可变区人源化设计

在分析抗体空间结构特点和残基间相互作用基础上,利用计算机辅助的分子设计和表面残基替换法进行鼠源抗体人源化设计。在抗体同源模建的基础上,利用序列分析和结构分析确定鼠源抗体可变区中外露的非人样差异残基,参考分子内、间氢键相互作用,最终选定将要突变的残基位点。使用这种方法对一株抗人肝癌细胞的单克隆抗体HAb18进行了人源化改造设计,并将候选位点分为三类,以便于在实验中依次突变,寻找降低鼠源抗体免疫原性和保持其生物功能之间适宜的平衡点。结果表明表面重塑方法可以有效地减少抗体人源化设计中CDR空间构象的变化,维持抗体生物活性,为试验提供有力的理论依据。     

基于人工免疫算法的电网负荷预测模型的研究

为了克服传统神经网络预测方法在网络结构设计和收敛效果等方面存在的缺陷。提出了一种进行电力系统负荷预测的新算法———人工免疫算法。该算法是根据高等动物免疫系统的机理而设计的,将目标函数和一部分不等式约束条件作为抗原,将搜索空间的解作为抗体,依据抗原与抗体的结合力以及抗体之间的结合力对解进行选择,通过抗体之间的相互激励作用提高了最优点附近的搜索效率,通过记忆细胞对抗体的抑制作用有效地摆脱局部最优点。应用该模型于阜新地区负荷预测的实例中,结果表明,该模型与传统的神经网络预测方法相比具有较强的自适应能力和较好的效果。     

Monoclonal antibody production by receptor-mediated electrically induced cell fusion

Fusion of myeloma cells and B lymphocytes to form hybridomas which produce monoclonal antibodies has been a major advance, but the poor efficiency and randomness of viral or polyethylene glycol fusion techniques generally gives poor yields of specific, high affinity antibodies. High voltage electrical fields with dielectrophoresis to ensure cell alignment can fuse a limited number of cells under direct microscopic examination, but it is not possible to identify B-cells destined to secrete relevant antibodies. However, B-cells express, on their surface, antigen receptor immunoglobulins of the same antigenic specificity as the secreted antibodies. Binding of antigen to surface immunoglobulins stimulates proliferation and differentiation of B-cells into plasma cells. Here we report the use of the selective, high affinity interaction of antigen with surface immunoglobulins on B-cells to facilitate a close adherence to myeloma cells. The antigen, covalently conjugated to avidin, binds to the surface immunoglobulins on B-cells. This B-cell-antigen-avidin complex binds to biotin covalently attached to the surface of myeloma cells. An intense electric field across a bulk cell suspension then produces selective fusion of cells in contact, that is, of myeloma cells with B-cells which make the appropriate antibody. We have used this technique with several antigens, and all resultant hybridomas secrete appropriate antibodies with very high affinity.     

植物表达抗体的研究进展

利用转基因植物生产抗体是一个新兴的生物技术领域。这种技术将编码全抗体或抗体片段的基因导入植物，从而在植物中产生全抗体或抗体片段，获得的抗体能功能性地识别抗原并结合抗原。目前已有农杆菌介导转移法和基因枪法等多种转化技术用于将抗体基因导入植物细胞。利用植物表达抗体的一大优势是能大规模廉价生产免疫治疗用抗体。此外，植物抗体也可用于植物自身抗病，并能调节植物细胞代谢。本文主要就植物生产抗体的方法、抗体的表达及应用作一综述。     

Three-dimensional structure of a complex of antibody with influenza virus neuraminidase

The structure of a complex between influenza virus neuraminidase and an antibody displays features inconsistent with the inflexible 'lock and key' model of antigen-antibody binding. The structure of the antigen changes on binding, and that of the antibody may also change; the interaction therefore has some of the character of a handshake.     

Therapeutic potential of monovalent monoclonal antibodies

One therapeutic use for monoclonal antibody technology is the elimination of categories of unwanted cells by virtue of their distinct cell surface antigens. The efficiency of cell destruction by complement lysis or opsonization depends on a number of factors such as antibody specificity and isotype as well as certain properties of the target antigen. In some instances cells can escape destruction by redistributing and eventually losing the antigen-antibody complexes from their surface. This process, known as antigenic modulation, generally depends on bivalent antibody binding. Starting from the observation that rabbit antisera can be made more effective at killing tumour cells if they are first rendered univalent by limited proteolysis, we have now prepared a number of monovalent rat monoclonal antibodies to human cell-surface antigens. We find that these antibodies are no longer able to bring about modulation of their target antigens and have an enhanced facility for lysis with human complement. These special properties should greatly increase the therapeutic potential of monoclonal antibodies.     

Inhibition of antigen-induced T-cell clone proliferation by antigen-specific antibodies

Attempts to inhibit the recognition of soluble antigens by T lymphocytes using antibodies specific for the antigen in question have been uniformally unsuccessful, in contrast to the observed specific inhibition of antibody generation by B cells. One exception is the unique situation whereby anti-hapten antisera inhibit the T-cell proliferative responses observed when hapten-specific T lymphocytes or clones are cultured with hapten-derivatized cells or proteins. The inability to inhibit T-cell functions by antigen-specific antibodies has been interpreted in several ways: (1) T cells possess a different repertoire from B cells; (2) the antibodies tested recognize epitopes present on the native antigen, whereas T cells recognize non-native (processed) structures; (3) the antigenic determinant(s) recognized by T cells on the surface of antigen presenting cells are either not accessible to antibodies, or are present in low amounts. The development of antigen-specific T-cell clones and monoclonal antibodies both specific for the same antigenic determinants now allows this question to be investigated definitively. Here, we report for the first time the specific inhibition of antigen-induced T-cell clone proliferation by a monoclonal antibody directed against the relevant soluble protein antigen.