应用单克隆抗体对烟草花叶毒病抗原决定簇及其抗原型的分析研究

收集国内9省、市、自治区14个烟草花叶病毒(TMV)分离物,在隔离条件下繁殖毒源,以同一方法,相同条件提纯并定量。以20μg/ml 为起始浓度,作10~(-1),10~(-2)、10~(-3)、10~(-4)……10~(-9)9个稀释度(设健康寄主对照),分别与适宜浓度(通过逐一测定滴度而选定的)10个 TMV 单克隆抗体(McAb)〔设多克隆抗体、正常细胞腹水(SP2/0)对照〕进行 ELISA 试验,结果从14株分离 TMV 分离物中识别到8种抗原决定簇,并将这些 TMV 分离物划分为六个抗原型。这揭示了应用 McAb 区分 TMV 株系是一个很有希望的方法     

巨细胞病毒单克隆抗体的建立和应用

应用人巨细胞病毒(CMV)AD169株免疫BALB/C小鼠,取脾细胞与SP 2/0小鼠骨髓瘤细胞融合,得到4株(12H、3H、47C和6H)能分泌单克隆抗体(McAb)的杂交瘤细胞。其中47C株McAb(滴度≥512 000)经辣根过氧化物酶标记,能与细胞培养中的AD169株和Davis株CMV发生特异性核内染色反应。14份出现CMV典型病变的临床阳性标本中,12份呈明显的阳性反应,另2份为弱阳性反应,而对正常细胞及感染其它病毒的细胞均无染色反应,说明该McAb具有较好的特异性。用于鉴定分离的毒株,一天内可出结果,比中和试验快速、简便。     

抗甲3型(H3N2)流行性感冒病毒单克隆抗体的建立

将SP2/0 Ag小鼠骨髓瘤细胞与经蔗糖梯度离心纯化的甲3型流感病毒沪防/32/84(H3N2)免疫的BALB/C鼠脾细胞融合,经初步克隆获得了25个能稳定分泌抗甲3型流感病毒单克隆抗体(McAb)的杂交瘤细胞株,部份McAb的一些生物学性状如下。 经ELISA测定25个腹水McAb、除4株为10~(-4)外,其余滴度在10~(-5)～10~(-(?))间。此25个McAb中,16个为抗血凝素McAb,其中15个的血凝抑制效价在1:1,280～1:20,480,1个为1:320,在鸡胚内均有不同     

分泌抗芜菁花叶病毒株系特异性单克隆抗体杂交瘤细胞株的建立及抗体理化性质测定

用经芜菁花叶病毒(TuMV)免疫的BALB／c小鼠脾细胞与骨髓瘤细胞(Sp2／0－Agl4)融合,经3次克隆化培养和ELISA筛选,建立了5类分泌抗TuMV的单克隆抗体(McAb)的杂交瘤细胞株。其中4类分别对TuMV－CI株系、TuMV—C3株系、TuMV—C4株系和TuMV－c5株系具有特异性反应,第5类对TuMV 5个株系均有反应。以双抗体夹心ELISA和间接ELISA检测,上述McAb同CaMV、CMV、TMV、PVX和PVY等均不产生交叉反应,小鼠腹水McAb的滴度在1：256000--2048000之间,多为1：1024000,比常规多抗血清高400倍左右。对上述杂交瘤细胞系和McAb的生物学特性及理化性质进行了鉴定。用SDS—PAGE、Western—blotting对TuMV外壳蛋白亚基、McAb识别位点进行了分析,并就McAb区分TuMV株系进行了讨。     

抗甲型肝炎病毒单克隆抗体(抗-HAV McAb)的研究

应用细胞融合技术成功地建立了3株持续高效分泌抗-HAV McAb的杂交瘤细胞株(AG_3,AD_2和AE_8株),所得腹水中的抗-HAV滴度达1:128000～1:1024000。这3株McAb都能与粪便提取HAV和细胞培养HAV反应,且这种反应均能被甲肝病人恢复期血清阻断。这些McAb作用于HAV的不同抗原位点,其中AE_8 McAb具有中和HAV感染性的能性。这些McAb作包被抗体检测HAV时,所测的HAV滴度较人抗体作包被时测得的滴度高2～8倍。将AE_8 McAb标记酶后,用以检测Abbott药盒标化的50份甲肝IgM血清,49份结果一致,符合率98％。     

抗仙台病毒杂交瘤细胞株的建立及所分泌单克隆抗体的部份理化性质

将经纯化仙台病毒免疫的Balb/c小鼠脾细胞与Sp2/0小鼠骨髓瘤细胞融合,经5次克隆和1次亚克隆,筛选出两株(5个亚株)分泌抗仙台病毒单克隆抗体(McAb)的杂交瘤细胞系。在组织培养上清液中,它们的HAI滴度为1:10～1:160,ELISA滴度为1:256～1:512;在腹水中,HAI滴度为1:320～1:1 280,ELISA滴度为1:32 000～120 000,抗体均为IgM。染色体数目为90～110条。 用该McAb所做的补体结合、中和及被动免疫试验均呈阴性反应,用免疫酶方法对电泳转移所得到的     

用流行性出血热单克隆抗体对我国不同地区出血热病毒株的抗原分析

从全国17个省市自治区收集了自病人及各种动物分离到的40株流行性出血热病毒(EHFV),用出血热病毒单克隆抗体(McAb)进行抗原分析,发现大多数省市分离的EHFV株能与MA25-1 McAb起反应,但湖北、湖南分离的EHFV不与之起反应,而该McAb对EHFVA9株抗原的滴度为1/2560。实验还发现,湖北省内长江以北地区分离的EHFV毒株Q25、J173。WP43和从江南地区分离的EHFV毒株A24和HA1018在抗原性上也有明显不同。     

抗B型葡萄球菌肠毒素单克隆抗体的研制及其鉴定

本文用纯化B型葡萄球菌肠毒素(SEB)免疫Balb/c小鼠的脾细胞与SP2/O骨髓瘤细胞融合,经筛选及克隆化共获6株能稳定分泌抗SEB的单克隆抗体(McAb)杂交瘤细胞株。通过将以混合佐剂(降植烷 FIA的混合物)和杂交瘤细胞同时注射制备McAb腹水的方法与常规法相比较,不仅量多(多1.56～1.84倍),而且活性好(高1个数量级)。初步鉴定表明:6株McAb均属IgGl亚类;其培养上清和腹水的ELISA滴度分别为10~(-3)～10~(-4)和10~(-5)～10~(-8)。它们与SEA均不起反应,其中3株(Sl.B4和E7)与SECl有轻微交叉反应;都能被10μg/m的SEB完全阻断等。说明其免疫学活性及特异性均较良好。     

肾综合征出血热病毒血凝素的研究 Ⅰ.病毒血凝活性的初步研究

以不同宿主来源的肾综合征出血热病毒(HFRSV)感染乳鼠,取脑经蔗糖-丙酮法制备血凝素,对这些毒株的血凝活性和血凝抑制(下称血抑,HI)反应进行了初步研究比较。结果提示受试毒株间血凝活性高低不等,似与对乳鼠的致病毒力有一定关系,血凝素抗原与其他病毒的免疫血清无交叉反应,在血凝活性较高的几个毒株间血凝素抗原性差异不明显,并找到了一株血抑滴度较高的单克隆抗体(McAb)。     

番木瓜环斑病毒单克隆抗体的制备及在病毒分型上的初步应用

本试验是用番木瓜环斑病毒(Papaya ringspot virus, PRV)提纯制剂免疫的BALB/c小白鼠脾细胞与Sp~2/o-Ag14骨髓瘤细胞融合,获得三个能稳定传代并分泌抗番木瓜环斑病毒的单克隆抗体的杂交瘤细胞系。其中23H1 McAb的效价较高,用ELISA检测,腹水抗体效价高达1:76800,能被PRV兔抗血清所阻断。这3个杂交瘤细胞系产生的单抗与TMV和CMV无血清交叉反应。它们可把PRV四个毒株初步区分为三个血清型。     

The mechanism(s) of coat protein-mediated resistance against tobacco mosaic virus

The resistance of transgenic plants express genes encoding viral coat proteins to infection by the viruses from which the genes are derived was termed coat protein-mediated resistance (CP-MR) and has been demonstrated for a variety of virus/host combinations. The mechanism of CP-MR is perhaps best understood in the tobacco/TMV system. CP-MR against TMV requires accumulation of CP and does not seem to involve the induction of plant defense mechanisms. The resistance appears to be mainly based on the inhibition of virion disassembly in transgenic cells although there is evidence that a later step of infection is also affected. CP-MR of tobacco to TMV shares some features with classical cross-protection and with CP-MR in some, but not all other host/virus combinations.     

Tumor-Derived Microvesicles Induce,Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

Background

Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4⁺CD25^highFOXP3⁺ Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg.

Methodology/Principal Findings

TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4⁺CD25^neg T cells into CD4⁺CD25^highFOXP3⁺ Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-β1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-β1 and/or IL-10 significantly inhibited TMV ability to expand Treg.

Conclusions/Significance

This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.     

Suppression of chronic antigen-induced arthritis in rats by a monoclonal antibody against the T cell receptor alpha beta.

We have investigated a role for T cells in chronic antigen-induced arthritis in rats employing a monoclonal antibody (R73 mAb) against the T cell receptor alpha beta. Treatment with R73 mAb from the time of intra-articular antigenic challenge blocked completely the induction of chronic, but not acute ovalbumin-induced arthritis in sensitized rats. Histologically, treatment-controlled arthritic rats exhibited marked hyperplasia of synovial membrane with pronounced infiltration of inflammatory cells including alpha beta + T cells in the chronic phase of arthritis. In contrast, R73 mAb-treated rats had almost normal joint histology. Treatment with R73 mAb after onset of arthritis was also effective in suppressing the progression of chronic antigen-induced joint inflammation. The preventive and suppressive effects of the mAb on chronic antigen-induced arthritis were associated with marked depletion of alpha beta + T cells in peripheral blood. The DTH but not the humoral response to ovalbumin in sensitized rats was suppressed significantly by R73 mAb. Thus, alpha beta + T cells appear to have a central role in both induction and progression of chronic antigen-induced arthritis.     

大麦黄花叶病毒单克隆抗体的制备

大麦黄花叶病毒(Barley yellow mosaic virus,BaYMV)广泛分布于西欧、日本和我国长江中下游和东部沿海地区,严重危害了大麦生产,并呈现出不断蔓延扩大的趋势。该病毒经土壤禾谷多粘菌介体感染大麦后,病毒繁殖发病的状况,易受到大麦品种不同、环境温湿度等因素的影响,发病程度的差异很大,而且还发现带毒隐症的大麦品种,使大麦抗源筛选和大麦抗性品种的选育工作受到严重干扰,因此迫切需要建立一种快速、灵敏和准确的检测方法。目前,比较理想的方法是ELISA等免疫学技术。     

Immunohistochemical monitoring of metastatic colorectal carcinoma in patients treated with monoclonal antibodies (MAb 17-1A)

Summary A therapeutic trial using repeated doses of a mouse monoclonal antibody against the tumor-associated antigen (TAA) CO17-1A in metastatic colorectal carcinomas was carried out. Metastatic lesions sampled by repeated thick needle (1.2 mm) biopsies during therapy were examined immunohistochemically for the presence of various TAAs, mouse IgG, complement, and infiltrating leukocytes. The CO17-1A was consistently expressed in all cases along the basement membrane of tumor glands and could only be demonstrated on cryostat sections whereas the TAAs GICA19-9, GA73-3, and Br55-2 were also visualized in B₅-fixed paraffin-embedded biopsies. The CO17-1A and GA73-3 were predominantly present at the basal region in contrast to the GICA 19-9 and Br55-2 which were predominant at the luminal and the apical region of the tumor glands. Antigenic modulation was not seen either after 24–72 h or during prolonged treatment. In all cases the infused mouse IgG was detected, from 24 h after infusion up to 6–8 weeks, mainly along the basal region of tumor glands. In 13/14 posttreatment biopsies, complement factor C3 was found at the same sites as mouse IgG. In 6 out of 9 posttreatment biopsies an increase in mononuclear cells (monocytes, natural killer (NK) cells and/or T cells) was observed. Monocytes were close to the tumor cells whereas NK cells and T cells were predominantly scattered in the stroma.This study was supported by grants from the Cancer Society in Stockholm, the King Gustav Vth Jubilee Fund, The Swedish Cancer Society and the Karolinska Institute Foundations     

Tumor-specific idiotype vaccines. II. Analysis of the tumor-related network response induced by the tumor and by internal image antigens (Ab2 beta)

In this study the tumor-specific immuneresponse induced by irradiated tumor cells (L1210/GZL) and by anti-idiotype antibodies was analyzed. The anti-idiotype antibodies (Ab2) were made against the paratope of a monoclonal antitumor antibody (11C1) that recognizes a tumor-associated antigen which cross-reacts with the mouse mammary tumor virus-encoded envelope glycoprotein 52. Two Ab2, 2F10 and 3A4, induced idiotypes expressed by the monoclonal antitumor antibodies 11C1 and 2B2. Cytotoxic T cells, generated by immunization with irradiated tumor cells, lyse 2F10 and 3A4 hybridoma cells. Furthermore, immunization with Ab2 induces tumor-specific cytotoxic T lymphocytes. The frequency of tumor-reactive cytotoxic T lymphocyte was found to be similar in mice immunized with Ab2 or irradiated tumor cells when examined at the precursor level. However, only 2F10 induces protective immunity against the growth of L1210/GZL tumor cells. The depletion of a L3T4+ T cell population from 2F10 immune mice was found to increase the effectiveness of transferred T cells to induce inhibition of tumor growth. The inability of 3A4 to induce antitumor immunity could be correlated with the presence of a population of Lyt2+ regulatory T cells. Collectively, these results demonstrate the existence of a regulatory network controlling the expression of effective tumor immunity. Our results demonstrate that selection of binding site-related Ab2 may not be a sufficient criteria for the development of an idiotype vaccine. A better understanding of the regulatory interactions induced by anti-idiotypes is needed for the design of effective antitumor immunotherapy.     

The Fc receptor for IgG on human natural killer cells: phenotypic, functional, and comparative studies with monoclonal antibodies

We compare five monoclonal antibodies ( B73 .1, 3G8 , Leu- 11a , Leu- 11b , and VEP13 ) that react with natural killer (NK) cells and polymorphonuclear cells (PMN). We show that all of these antibodies are directed against and inhibit the functional properties of the receptor for the Fc portion of IgG (FcR). Modulation of the FcR on NK cells after reaction with immune complexes induces the disappearance of the antigen(s) recognized by each of the five antibodies. Conversely, the antibodies block binding of IgG-sensitized erythrocytes to the NK cells and PMN and inhibit their ability to mediate cytotoxicity against antibody-sensitized tumor target cells. By using two-color immunofluorescence techniques, we characterize directly the lymphocyte population recognized by these antibodies and show that it is a homogeneous subset that does not bear markers of either B or T cells, with the exception of the 33,000 dalton antigen characteristic of suppressor/cytotoxic T cells present in 20 to 50% of the cells, and the 45,000 dalton receptor for sheep erythrocytes present on 80 to 90% of the cells. The phenotype of the cells reacting with the monoclonal antibodies corresponds to that of NK cells. Cross-competition experiments indicate that these antibodies detect at least two distinct epitopes on FcR, one ( B73 .1) preferentially expressed on NK cells and one or more ( 3G8 /Leu- 11a /Leu- 11b / VEP13 ) preferentially expressed on PMN. The lack of reactivity of these antibodies with B cells suggests that human B cells bear a different FcR from that on NK cells and PMN.     

Monoclonal antibodies against the FhuA (TonA) protein of the Escherichia coli outer membrane

Abstract Outer membranes of Escherichia coli K-12 were used to isolate hybridoma cell lines that produce monoclonal antibodies against the FhuA (TonA) protein. Two monoclonal antibodies were obtained from independent immunization and fusion experiments. The antibodies belonged to the subclass IgG₁ and κ, and IgG_2b and κ, respectively. The latter antibody was purified by affinity chromatography on protein A-Sepharose. The culture supernatants of the hybridoma cell lines and the isolated antibody inhibited adsorption of the phages T5 and T1 to E. coli cells while binding of phage ø80, which also uses the FhuA protein as a receptor, remained unaffected. The specificity of the antibodies to the FhuA protein was supported by the prevention of killing of cells by colicin M and by the lack of inhibition of colicin B and of phage BG23. Transport of iron(III) as ferrichrome complex was not inhibited by the isolated antibody. However, partial competition with the adsorption of the phages T2, TuIb and T6 was observed which may indicate an organization of certain functional phage receptors into clusters.     

A DNA vaccine against cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) prevents tumor growth

Co-stimulatory signaling pathway triggered by the binding of B7.1/B7.2 (CD80/86) of antigen-presenting cells (APCs) to CD28 of T cells is required for optimal T-cell activation. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding with a greater affinity. Ipilimumab, a monoclonal antibody against CTLA-4, has shown positive efficacy in a pivotal clinical trial for the treatment of metastatic melanoma and was approved by FDA. However, the cost of monoclonal antibody-based therapeutics might limit the number of patients treated. To develop a novel therapeutics specifically targeting CTLA-4, we constructed a DNA vaccine by cloning the sequence of CTLA-4 fused with a transmembrane domain sequence of placental alkaline phosphatase (PLAP) into a mammalian expression plasmid, pVAC-1. Immunization with the resulting construct, pVAC-1-hCTLA-4, elicited antibody specific to human CTLA-4 with cross reactivity to murine CTLA-4, which was sufficient for inhibiting B16F10 tumor growth in c57BL/6 mice in the absence of measurable toxicity. Coupling liposome with pVAC-1-mCTLA-4 could break tolerance to self-antigen in BALB/c mice and induce potent immunity against murine CTLA-4, and suppress growth of subcutaneous renal cell carcinoma (Renca).