合成人IgE肽抗血清抑制I型变态反应的实验研究

作者设计合成了５个人ＩｇＥ多肽片段，分别交联载体蛋白后免疫小鼠，诱生的抗血清２个在ＥＬＩＳＡ中对人与鼠ＩｇＥ分子有交叉反应，３个无反应，５个抗血清均显示有中等程度的被动皮肤过敏反应（ＰＣＡ）抑制作用，被测试的２个抗血清在大鼠肥大细胞被协致敏试验中也显示了抑制作用，研究结果初步表明，采用人ＩｇＥ分子受体结合部位的适当残基序列合成肽疫苗，免疫动物所诱导的抗体可抑制１型变态反应。     

恶性疟原虫杂合抗原基因的表达及其产物免疫功能的研究

化学合成的人恶性疟原虫杂合多肽抗原 AB 基因,编码子孢子 CSP重复序列 NANP、CSPTh2R和红内期spf35.1、spf83.1、spf55.1、spf83.18、pf155/RESA-5’端重复区共7个不同免疫功能表位,与大肠杆菌质粒pWR450-1重组,构建成pWR450-1/AB表达载体。在乳糖操纵子调控下以β-半乳糖苷酶-恶性疟原虫杂合多肽抗原融合蛋白形式,在大肠杆菌中高效表达。表达产物经SDS-PAGE电泳分离纯化后,加福氏佐剂免疫家兔,诱导产生较高滴度的抗血清。抗血清对人恶性疟原虫体外生长有明显的抑制作用,24h抑制率为65.92％;72h的抑制率为72.63％。对照血清无抑制作用。分离纯化的表达产物能与鼠抗恶性疟原虫和疟疾患者抗血清起免疫反应。这些结果表明合成基因的表达产物具有较好的免疫原性。     

重组人bFGF的原核表达及其高效价抗血清的制备

目的 以重组人碱性成纤维生长因子为免疫原，制备高效价抗hbFGF抗血清。方法通过PCR方法改造5’编码区的12个密码子，构建hbFGF’原核表达载体并在大肠杆菌(E．coli)中表达，以纯化的hbFGF、免疫新西兰兔，制备高效价抗血清，用于重组hbFGF、的免疫印迹分析。结果经过改造的hbFGF基因在E．coli中获得较高水平表达。从可溶性部分纯化得到纯度95％以上的重组hbFGF，以该重组蛋白免疫兔子，在二次加强后以间接ELISA检测抗血清效价可达1：512000。免疫印迹分析显示该抗血清与E．coli中表达的重组hbFGF、和标准hbFGF、均有特异性反应，但与某些细菌蛋白存在弱交叉反应，经E．coli菌体蛋白吸附的抗血清，与菌体蛋白的弱交叉反应消失。结论以纯化的重组hbFGF为免疫原制备了高效价的特异性抗血清，经菌体蛋白吸附可消除存在的交叉反应性。     

高活力人降钙素类似物vhCT的免疫原性研究

本研究用固相多肽合成法合成人降钙素类似物vhCT ,产物纯度达到等电聚焦均一 ,质谱测定分子量精确。生物活性为天然人降钙素的 2 0～ 2 5倍。用放射免疫法测定vhCT能与识别天然人降钙素的抗体起反应 ,产生IgG抗体的免疫原性随偶联的不同载体而有差异。但在BALB/C5 7小鼠体内不引起IgE增高。提示vhCT不产生IgE介导的过敏反应 ,具有药物应用前景。     

兔抗人DR5抗血清诱导Jurkat细胞凋亡

制备兔抗人sDR5抗血清,检测它对Jurkat细胞的生长抑制和凋亡诱导作用。采用本室制备的sDR5免疫新西兰白兔,制备兔抗人sDR5抗血清,用ELISA法测定抗sDR5抗血清效价及抗血清的特异性。MTT试验分析它对Jurkat细胞生长抑制影响,倒置光显微镜和荧光显微镜观察抗sDR5抗血清对Jurkat细胞形态的影响,用AnnexinV/PI双染试剂盒检测Jurkat细胞凋亡率,琼脂糖凝胶电泳检测Jurkat细胞中DNA的片断化。结果:获得了高效价特异性兔抗人sDR5抗血清。兔抗人sDR5抗血清对Jurkat细胞具有显著的细胞生长抑制作用,并呈剂量依赖性。兔抗人sDR5抗血清处理后,Jurkat细胞可出现典型的细胞凋亡的形态特征:细胞膜皱缩,出泡,染色质浓缩,形成凋亡小体等。流式细胞术结果显示:兔抗人sDR5血清1/80、1/160作用Jurkat细胞2 h,细胞凋亡率分别为54.98%和34.13%。兔抗人sDR5抗血清可导致Jurkat细胞中的DNA片段化。本室制备的兔抗人sDR5抗血清能抑制Jurkat细胞生长和诱导Jurkat细胞凋亡。     

IgE在免疫损伤大鼠弓形虫病中的保护性作用

人工感染刚地弓形虫后,免疫损伤的Nu/Nu大鼠很快死亡。被动转动感染弓形虫28天的Fischer大鼠血清后,其存活期明显延长,但转移灭活IgE的血清则无效,说明这种抗感染的保护性作用与IgE有关。免疫功能正常的大鼠感染弓形虫后7天,可于血清中检出特异性IgE,并于21至28天达高峰。再次感染后5天,出现第2个     

CD23研究进展

本文从CD23与IgE、CD23与其受体,与CD23有关的信号传导和基因调控等几个方面介绍了CD23研究的最新进展。CD23,即IgE的低亲和力受体(FceRⅡ),是一个45KD的糖蛋白。它是唯一的不属于Ig超家族的Fc受体,与C型植物血凝素有很多相似之处,这个家族包括选择素如MEL14、LAM1、ELAM1、CD62等以及其它各种粘附分子。CD23分子最早在1975年由Lawrence等发现,并于1986年由Kikutani等人克隆出来。CD23和其它生物分子的相互作用引发一系列生物活性,比如细胞与细胞粘附、调节IgE合成、抗原呈递、早期T细胞分化、防止生发中心B细胞凋亡、炎症反应调节、肥大细胞及嗜碱性粒细胞释放组胺、对寄生虫的免疫攻击、调节HIV1的扩增等等。本文从以下几个角度对CD23研究的新进展作一综述。     

特异性过敏原TBt多克隆抗体的制备与鉴定

目的:制备特异性苦荞主要过敏原TBt的多克隆抗体,为深入研究TBt分子中各结构域的功能奠定基础.方法:通过盐析,离子交换层析,分子筛层析等方法从苦荞种子中纯化出过敏蛋白TBt,免疫新西兰兔子,制备多克隆抗体.用间接ELISA、Western blot检测该抗体的效价和特异性；竞争ELISA研究IgG抗体对TBt过敏患者血清IgE的抑制作用.结果:间接ELISA法检测所制备抗体的效价达1∶256000左右；Western blot显示该抗体能与TBt蛋白特异结合；竞争ELISA表明制备的IgG抗体能够特异性抑制养麦过敏患者血清1gE与过敏蛋白的结合.结论:制备的TBt多抗血清具有较高的效价和良好的特异性,该多克隆抗体可用于TBt的免疫活性鉴定,为下一步研究该蛋白的分子特征及免疫治疗奠定了基础.     

fgl2凝血酶原酶多肽抗体的制备和应用

目的　利用人工合成多肽制备针对人源、鼠源fgl2凝血酶原酶 (hfgl2、mfgl2 )的特异性多克隆抗体 ,以期用于与fgl2表达异常密切相关的疾病的研究和诊治。方法　根据mfgl2、hfgl2基因编码的氨基酸序列合成多肽 3(Pep3)和多肽 4 (Pep4 ) ,并用化学方法与匙孔血蓝蛋白 (KLH)连接 ,纯品Pep3 KLH和Pep4 KLH免疫动物 ,所制备的抗血清用ELISA、Westernblot和前凝血质活性 (PCA)实验鉴定 ,并采用免疫组化法应用于乙型肝炎患者肝组织中hfgl2的检测。结果　ELISA检测表明 ,所制备的抗血清可分别与Pep3和Pep4发生特异性免疫反应 ;Westernblot结果显示 ,抗血清可识别MHV 3感染BALB cJ小鼠腹腔巨噬细胞特异性条带 ,相对分子质量 (Mr)为 6 5× 10 3;PCA实验提示 ,抗血清具有中和mfgl2分子酶活性的作用。对病毒性乙型肝炎患者肝组织免疫组织化学染色发现hfgl2仅在重型乙型肝炎患者高表达 ,而在慢性乙型肝炎和肝硬化患者的肝组织中无表达。结论　所制备的fgl2的多肽抗体 (多克隆抗体 )可识别mfgl2和hfgl2分子 ,具有与抗原特异性结合、中和其酶活性的特征 ,为深入研究这一新发现的分子提供了有力的科学手段。     

抗人IgE血清的制备及其在ELISA中的应用——Ⅱ.羊抗人IgE血清的制备及用ELISA法测定人血清总IgE水平

本文介绍羊抗人 IgE 血清制备、纯化的方法学。用纯度较高的 IgE 骨髓瘤蛋白抗原,采用淋巴结微量免疫法免疫绵羊,制备出特异性羊抗人 IgE 血清。用琼脂双扩散法和免疫电泳祛分别鉴定其效价和纯度。用45%饱和硫酸铵盐析法进行粗提,再经 DEAE_(82)-纤维素离子交换柱层析,利用阶段洗脱法得以进一步纯化,制备出纯度较高的羊抗人 IgE 的特异性抗体(IgG 部分)。为了鉴定此特异性抗体的效价,应用 ELISA 测定人血清总 IgE 水平。本文对国外进口同类抗血清制品以及相应的辣根过氧化物酶结合物进行了比较。     

Influence of the medium on the heat and acid denaturation of IgE

The denaturation of IgE immunoglobulin induced by heating at 56 degrees C or by treatment at low pH is inhibited in the presence of high concentrations of salts or hexoses. Between 50 and 100% of the IgE anaphylactic activity (PCA) of rat and mouse antisera is recovered after heating at 56 degrees C for 1,5 or 5 h, respectively, in 1 M MgSO4 or 2 M glucose, mannose or fructose. Anaphylactic activity of IgE monoclonal anti-DNP mouse antibody is equally preserved. The specific antigenic determinants of human and rat IgE myeloma proteins are also thermostable in these conditions. The addition of MgSO4 or glucose protects IgE anaphylactic antibodies against denaturation at pH 2. It is suggested that IgE denaturation is the consequence of interactions between molecules of immunoglobulin and that such interactions are diminished by steric hindrance in a medium containing high concentrations of ions or hexose molecules.     

Passive cutaneous anaphylaxis inhibition: evidence for heterogeneity in IgE mast cell interaction.

Recent evidence suggests that IgE molecules are heterogeneous with respect to ability to compete with IgE myeloma for sensitization of histamine release from chopped human lung and ability to passively sensitize human basophils for antigen-induced histamine release. These observations prompted further investigation of the possibility that there exists a functional heterogeneity in the IgE molecules with respect to mast-cell binding properties. Using eight different purified rat IgE myeloma proteins, we found that they differ in their ability to inhibit the passive cutaneous anaphylaxis (PCA) reaction of mouse reaginic antisera. This suggests that IgE molecules differ in their ability to bind to mast cell receptors. Since maximal inhibition of different mouse reaginic antisera and mouse IgE hybridomas is achieved with different IgE myelomas, there may exist a functional heterogeneity in mast-cell binding receptors as well.     

Novel IgE peptide-based vaccine prevents the increase of IgE and down-regulates elevated IgE in rodents

BACKGROUND: Immunotherapy with anti-IgE antibodies for treatment of allergy is promising but a short half-life and extremely high cost limit its application. OBJECTIVE: We sought to develop IgE vaccines that induce longer-lasting auto-antibodies to neutralize self-IgE as an alternative therapy. METHODS: The vaccine was made by conjugating three synthetic peptides corresponding to human IgE receptor-binding sites to a carrier, hepatitis B surface antigen. To test the immunogenicity of the vaccine, rats were immunized with the vaccine or hepatitis B surface antigen as control. Serum IgG titres to human IgE and the IgE of other species were measured. The inhibition by rat antisera of the binding of human IgE to its receptor was assessed by ELISA, flow cytometry analysis, and passive cutaneous anaphylaxis (PCA), and its ability to recognize receptor-bound IgE was examined. The in vivo effect of the vaccine was evaluated in trichosanthin-sensitized mice and rats. In the preventative study, vaccination started before sensitization commenced, while in the treatment study, vaccination started after sensitization. Sensitized mice and rats receiving injections of the carrier served as controls. Trichosanthin-specific IgE was measured using PCA. RESULTS: Sera from vaccine-immunized rats contained high titre antibodies that reacted with soluble and plate-bound but not with receptor-bound human IgE; they also reacted with mouse, rat, and dog IgE. Furthermore, the sera inhibited the binding of human IgE to its receptor in a dose-dependent manner. In preventative and treatment studies, serum trichosanthin-specific IgE levels were significantly reduced in vaccinated groups compared with controls. CONCLUSION: Antibodies against self-IgE can be induced by IgE peptide-based vaccines, which are effective in preventing the increase of IgE and in down-regulating IgE in sensitized animals.     

Brown Norway rat ovalbumin-specific immunoglobulin E antibodies increase the human basophil expression of CD63 marker

Anaphylactic shock is an immunoglobulin E (IgE)-dependent hypersensitivity. Biological tests like leucocyte histamine release (LHR) and human basophil activation (HBA), frequently used in human allergy, reflect both the amount of IgE fixed on cells and the cellular reactivity. To assess whether serum-specific IgE from Brown Norway (BN) rats prepared for ovalbumin (OVA)-induced anaphylactic shocks can activate human basophils which has a potential interest in experimental allergy: such a test could rapidly assert an IgE sensitization in laboratory animals genetically T-helper 2 (Th2)-predisposed. Rats (n = 39) were immunized three times (day 0, day 5 and day 21) with OVA injected subcutaneously. One week after the third immunization, a shock was induced with an intravenous (i.v.) bolus of OVA. Sensitization was assessed by passive cutaneous anaphylaxis (PCA) test and dosages of serum IgE antibodies anti-OVA by enzyme-linked immunosorbent assay. Blood basophils were counted before and during the shock. Before the shock induction (at day 21), an LHR test was performed on rat blood, and human basophils were sensitized with rat sera. HBA was demonstrated by the increase in the percentage of cells expressing CD63 antigen membrane, measured by flow cytometry. Twenty-one days after the first subcutaneous (s.c.) immunization, the rat serum induced a significant HBA. HBA was observed neither with the same serum previously heated nor with the serum from nonimmunized rats (NIRs). OVA-specific IgEs were significantly increased in immunized rat (IR) serum. The PCA test was negative when the serum was previously heated (56 degrees C). We never observed any circulating basophils, and LHR test was negative. After OVA i.v. administration, all IRs died rapidly. HBA testing strongly suggests a mediation by specific IgE in the increase of CD63 in BN rats. Thus, HBA test seems useful in assessing whether an experimental allergy was induced in animals genetically predisposed to an immune response, Th2-mediated, like BN rat. We also conclude that rat basophil activation does not participate in the histamine release during anaphylactic shock in sensitized BN rats.     

The possibilities of predicting allergic effects of new antirheumatics

The new antirheumatic drugs Benzofenac and Flobufen were assessed for their possible influence on various immunological parameters. They were shown to enhance passive cutaneous anaphylaxis (PCA) to ovalbumin in rats and to suppress the numbers of helper and suppressor T-lymphocytes in mice. The effect of Benzofenac in the latter system was found to be more pronounced. The drugs had no effects in any of the other test systems, including the development of delayed hypersensitivity, mitogen-induced lymphocyte proliferationin vitro and the production of antisera (as tested by PCA).     

Properties of mouse homocytotropic and heterocytotropic antibodies.

Mouse antisera were analyzed for the presence of homocytotropic and heterocytotropic antibodies. Two distinct populations of antibodies were detected by the homologous passive cutaneous anaphylaxis (PCA) reaction. The first was active 2 hr after injection, heat-stable, and partially reactive with antisera to mouse IgG1. The second was active 48 hr after injection, was heat-labile, and probably belonged to the IgE class of mouse immunoglobulins. In addition, it was demonstrated that there were also two antibodies active in the heterologous PCA reaction in rats, a heat-stable antibody and a heat=labile antibody. Contrary to results obtained with homocytotropic antibodies, none of the heterocytotropic antibodies detected reacted with antisera to mouse IgG1 or IgG2. These studies suggest that in addition to IgE there may exist another heterocytotropic antibody in mouse antisera and that caution should be employed when using the 2-hr PCA reaction in the rat as a sole criterion for detection of mouse IgE.     

Effect of Trichinella spiralis infection on passive cutaneous anaphylaxis in mice.

Infection of CFW mice with Trichinella spiralis induced a state of relative unresponsiveness to passive cutaneous anaphylaxis (PCA) induced with hen egg albumin and its corresponding antibodies. The unresponsiveness was to PCA produced either with immunoglobulin G1 (IgG1) or IgE type of antibodies, but was more pronounced with the latter. As few as 25 larvae given by stomach tube 20 days before induced this resistance, although 400 larvae induced a greater resistance. When 400 to 600 larvae were fed to mice, the refractoriness of these mice to PCA was noticed 15 days later. The sera of infected mice had the ability to inhibit mainly PCA induced by IgE. This inhibitory property of sera from infected mice was more pronounced 35 days after infection than 10 months later, when only weak inhibitory activity was detected. Purified rat IgE inhibited the PCA reactions induced in both mice and rats with mouse IgE-type antibody. At high concentrations, evidence of inhibition of the IgG1-induced PCA in mice was also obtained. We believe that the relative unresponsiveness of infected mice is due to an increase in production of IgE which competitively blocks the mast cell sites for other IgE molecules.     

Induction of an auto-anti-IgE response in rats. IV. Effects on mast cell degranulation.

Induction of an auto-anti-IgE (auto-aIgE) response in the rat inhibits both total and specific IgE production and alters the distribution of mast cell (MC) subpopulations identified by differential Alcian blue/safranin staining. We have extended these observations by characterizing the auto-aIgE antibodies and determining their effects on MC degranulation in vitro and in vivo. An auto-aIgE response was induced in bacillus Calmette-Guérin (BCG)-primed rats by injecting a conjugate of highly purified rat IgE myeloma (IR2) coupled to tuberculin-derived purified protein derivative (PPD). Anti-IgE autoantibodies were almost exclusively IgG2a. The intradermal injection of auto-aIgE into untreated rats induced local MC degranulation as shown by a strong immediate skin response. Histologically there was evidence of significant degranulation of safranin staining connective tissue MC (SMC) in the skin but not of the Alcian blue staining MC (ABMC) in the sub-epidermal region. The induced degranulation was epsilon-chain specific; immunopurified anti-idiotypic antibodies raised to the IgE IR2 myeloma had no MC degranulating activity. When administered locally, auto-aIgE inhibited a subsequent passive cutaneous anaphylaxis (PCA) response elicited by anti-ovalbumin IgE. In addition, the PCA response was significantly decreased in animals with an ongoing auto-aIgE response. Immunopurified auto-aIgE also induced histamine release in vitro from rat peritoneal MC. These results are discussed in the context of naturally occurring autoantibodies to IgE present in patients with allergic disease.     

Detection and characterization of polymers in cephalothin by passive cutaneous anaphylaxis in mice

Immunization of BALB/c mice by repeated injections of cephalothin (CET) - Ascaris suum extract conjugate resulted in formation of IgE antibodies, which were able to sensitize syngeneic animals for passive cutaneous anaphylaxis (PCA). By means of high-pressure liquid chromatography, a fraction with extremely high PCA-eliciting activity, but without appreciable antimicrobial activity, was isolated from the CET preparation. Physicochemical analyses of the fraction identified the major component of polymer impurities as being a proteinaceous complex with a molecular weight of 6,580. Very little cross-reactivity of CET and potassium benzyl penicillin (PcG) was noted when these antibiotics were used as the challenge antigens for PCA induced by corresponding murine antisera. The results of the inhibition studies indicated, however, that at least two antigens were involved in the PCA induced by anti-CET antibodies, one strictly specific for CET and another shared by PcG. Evidence was also presented that the nucleus structure and acyl side-chain structure of CET play the major role in the PCA elicited by the challenge with CET and its polymer, respectively.     

Separation of mouse homocytotropic antibodies by biological screening

An attempt was made to separate mouse γ₁ antibody from mouse reaginic antibody by injecting mouse antiserum containing both antibodies into normal mice and then at 6 and 24 hours bleeding the animals and testing their sera for passive cutaneous anaphylaxis (PCA) activity. This was termed biological screening. The 2-hour homologous PCA activity was used as a measurement of mouse γ₁ and the rat PCA activity of the mouse antisera was used as a measurement of mouse reaginic antibody. These experiments showed that in vivo screening of mouse antisera containing both mouse and rat PCA activity results in removal of the rat PCA activity of these sera whereas the mouse PCA activity remains practically unchanged. It is concluded that a separation of the mouse serum PCA activity due to γ₁ antibody from that due to mouse reaginic antibody can be achieved by biological screening. Screening experiments using very early mouse antisera collected 8 days after a single antigenic stimulation resulted in the simultaneous disappearance of both homologous and heterologous PCA activity. Heating of these very early antisera resulted in complete inactivation of heterologous PCA activity and almost complete inactivation of homologous PCA activity. Absorption of these same antisera with rabbit anti-mouse γ₁ caused no change in homologous or heterologous PCA activity. It is suggested that the PCA activity of the very early antisera is due almost entirely to mouse reaginic antibody.