抗人B7-H1单克隆抗体的制备和鉴定

目的:采用杂交瘤技术制备抗人B7-H1单克隆抗体,并对其进行鉴定。方法:经抗原免疫的小鼠脾细胞与小鼠骨髓瘤细胞以常规方法融合;用间接ELISA法筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法获得稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注射进小鼠腹腔后制备腹水;纯化腹水中的单克隆抗体并对其亚型进行鉴定;用间接ELISA法测抗体效价;将肺癌组织制成石蜡切片,用抗人B7-H1抗体进行免疫组化染色。结果:获得1株稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株,所分泌的单抗类型为IgG1;抗体效价为1×108,纯化后的抗体含量为6.76g/L;免疫组化实验中,单抗可与肺癌组织表面的B7-H1蛋白特异地结合。结论:制备了人B7-H1单克隆抗体,为B7-H1检测试剂盒的研制奠定了基础。     

人凝血因子Ⅶ单克隆抗体的制备与鉴定

目的：制备抗人凝血因子Ⅶ单克隆抗体并鉴定其特性。方法：应用杂交瘤融合技术,以重组人凝血因子Ⅶ为抗原免疫BALB／c小鼠;取免疫小鼠的脾细胞与Sp2／0骨髓瘤细胞融合,经间接ELISA法筛选、融合细胞有限稀释法克隆、克隆化杂交瘤细胞株的亚类鉴定等方法筛选出单克隆抗体杂交瘤细胞株,并对单克隆抗体的特异性进行鉴定;用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行单抗的纯化。结果：获得了3株可稳定分泌单克隆抗体的杂交瘤细胞3E8、3D2和1C5,诱生的腹水效价分别为1：1×10^7、1：1×10^6和1：1×10^6;亚类鉴定表明388为IgG2a,其余2株均为IgGl;特异性鉴定显示它们与多种血浆蛋白均无交叉反应,表明单抗是特异的;经过亲和层析,获得了纯化的单抗。结论：获得了特异性的人凝血因子Ⅶ单克隆抗体,为建立人凝血因子Ⅶ检测及纯化方法奠定了基础。     

抗猪PD-L1单克隆抗体的制备与其生物学特性鉴定

本研究旨在制备抗猪PD-L1单克隆抗体(monoclonal antibody,m Ab),有助于阻断猪PD-1/PD-L1通路逆转免疫功能。免疫原为猪PD-L1胞外区重组蛋白,雌性BALB/c鼠为免疫动物,用淋巴细胞杂交瘤技术将鼠骨髓瘤细胞NS0和免疫BALB/c鼠脾细胞融合,ELISA法筛选及多次克隆化培养,筛选抗猪PD-L1 mAb的杂交瘤细胞株。成功制备1株能稳定分泌抗猪PD-L1的mAb的杂交瘤细胞株3B5,Ig亚型为IgG1,细胞上清和腹水效价分别为1:1×2~(10)和1:1.024×10~5,ELISA和Western-blotting结果表明该株单抗能特异性识别猪重组PD-L1蛋白,流式细胞术结果表明该单抗可以与猪PBMC上PD-L1蛋白有效结合,成功制备了1株分泌抗猪PD-L1单克隆抗体的杂交瘤细胞株3B5,为检测猪PD-L1蛋白表达水平及猪PD-1通路在猪传染性疾病中的致病机制提供有力的检测工具。     

蚕豆萎蔫病毒单克隆抗体制备及检测应用

:用蚕豆萎蔫病毒(BBWV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得6株能稳定传代并分泌抗BBWV单克隆抗体(Mab)的杂交瘤细胞株,单抗腹水ELISA滴度为1:320000～1:640000,各单抗抗体类型均为IgG1。6株单抗与BBWV不同分离物均有反应,而与其它植物病毒无交叉反应。经Westernblot印迹分析表明,此6株单克隆抗体均是针对BBWV447kD的外壳蛋白大亚基的特异性抗体。这是国内外首次报道获得BBWV单克隆抗体     

禽网状内皮组织增殖病病毒gp90蛋白单克隆抗体的制备及其识别区分析

为了制备禽网状内皮组织增殖病病毒(REV)gp90蛋白的单克隆抗体,应用His-gp90融合蛋白免疫BALB/c小鼠,取免疫鼠的脾细胞与骨髓瘤细胞(SP2/0)进行融合,经过筛选、3次亚克隆后获得3株稳定分泌抗REV-gp90蛋白的单克隆抗体杂交瘤细胞株,分别命名为3G5-B8、3G5-A10和1G12。经间接ELISA(Enzyme-linked immunosorbent assay)方法检测,单克隆抗体的亲和力解离常数(Kd)分别为6.483×10–10、4.844×10–10和9.330×10–10,3株单抗的亚型分别为Ig G1、Ig G1和Ig G2b。经Western blotting和间接免疫荧光实验检测,3株单抗均能识别REV感染DF-1细胞后产生的gp90蛋白。以Western blotting方法利用单抗检测不同截短的gp90蛋白,初步确定3G5-B8和3G5-A10 2株单抗抗原识别区均位于gp90蛋白第200-245位氨基酸,而1G12株单抗识别区包含第230-235位氨基酸。这些单抗为REV的诊断和致病机理研究奠定了基础。     

抗传染性法氏囊病病毒VP5蛋白单克隆抗体的制备与初步应用

原核表达的IBDVGx-VP5蛋白经纯化后免疫8周龄的BALB/c雌性小鼠,三次基础免疫后,融合前加强免疫,取脾细胞在PEG(MW1500)的作用下与SP2/0小鼠骨髓瘤细胞融合,经过三次亚克隆筛选,获得稳定分泌抗VP5蛋白的杂交瘤细胞,分别命名为4B4、6D12、3E8,以三株杂交瘤细胞制备的腹水ELISA效价分别为5×104、3.5×104、3×104,特异性实验表明三株单抗能与IBDVGt株反应。以单抗介导的间接免疫荧光检测表达Gt-VP5的VeroE6细胞,可以见到特异的荧光,能做为特异性的检测VP5蛋白的工具,为今后IBDVVP5蛋白的研究打下基础。     

口蹄疫病毒单克隆抗体的制备及检测应用

用纯化的口蹄疫病毒(Footandmouthdiseasevirus,FMDV)免疫BALB/C小鼠,将免疫鼠的脾细胞与SP2/0骨髓瘤细胞融合,采用有限稀释法进行克隆,经筛选获得多株能稳定分泌抗FMDV单抗的杂交瘤细胞株。选择其中一株(2G12)用于下列实验,其细胞培养上清液的效价是1:256,腹水效价是1:1280;以自行制备的兔抗FMDV高免血清IgG为捕获抗体包被酶联免疫吸附试验微量反应板,以单抗2G12为第二抗体,建立了快速检测FMDV抗原的双抗体夹心ELISA,该方法能检出90ng病毒,而且只与FMDV发生特异性反应,与猪瘟病毒(HCV)、猪蓝耳病病毒(PRRSV)、伪狂犬病毒(PRV)、猪细小病毒(PPV)和乙脑病毒(JEV)均不发生反应。本研究为检测口蹄疫病毒抗原提供了灵敏和特异的方法。     

抗人肝素酶单克隆抗体的制备与鉴定

目的:肝素酶在白细胞游走和恶性肿瘤转移的过程中发挥重要作用,肝素酶抗体的制备对于自身免疫病和肿瘤的良恶性鉴别诊断具有重要意义。制备抗人肝素酶单克隆抗体,用于肝素酶的研究及临床恶性肿瘤的鉴别诊断。方法:通过杂交瘤技术将分泌抗人肝素酶单抗的小鼠B细胞与小鼠骨髓瘤细胞Sp2/0融合,获得稳定分泌抗人肝素酶单抗的杂交瘤细胞;用有限稀释法获得单克隆,以重组人肝素酶及含肝素酶的血小板裂解液对抗体进行Western印迹检测。结果:Western印迹结果显示制备的单抗与人肝素酶具有特异性免疫识别特性。结论:获得了能够特异性免疫识别人肝素酶的分泌性抗人肝素酶单克隆抗体。     

甲型H1N1流感病毒血凝素蛋白单抗杂交瘤细胞株的制备与初步鉴定

目的:建立具有高特异、高效价的甲型H1N1流感病毒血凝素蛋白(HA)单抗的杂交瘤细胞株。方法:以纯化的昆虫杆状病毒表达的甲型H1N1流感病毒HA蛋白为免疫原免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,通过有限稀释法筛选阳性克隆,经ELISA和Western blot分析单抗的特性和特异性。结果:获得6株甲型H1N1流感HA抗原特异单克隆抗体杂交瘤细胞株,抗原肽库ELISA检测结果表明其中3株(1E12,3F12,1C11)单抗只与甲型H1N1流感HA抗原肽库反应,不与H5N1病毒HA抗原肽库反应;Western blot分析表明,单抗1B3只特异识别甲型H1N1流感HA抗原,而与其他季节性甲流病毒(H1,H3)及人禽流感H5N1病毒不反应。结论:所获杂交瘤细胞株特异性强,效价高,分泌抗体性能稳定,为分析甲型H1N1流感病毒抗原性位点、建立诊断试剂奠定了基础。     

鸡传染性贫血病毒VP3蛋白单克隆抗体的制备

以原核表达的鸡传染性贫血病毒(CIAV)VP3蛋白作为免疫原,免疫BALB/c小鼠,三次免疫后,取免疫小鼠脾细胞与SP2/0细胞融合,融合阳性细胞用间接ELISA方法检测,阳性细胞株经三代克隆纯化后,获得三株能稳定分泌抗CIAV VP3蛋白单克隆抗体的杂交瘤细胞株,分别命名为AG2、BC11、FG1。三株杂交瘤细胞株产生的细胞培养液上清和小鼠腹水的ELISA效价分别为2.5ⅹ102、1.5ⅹ102、1ⅹ102和2ⅹ106、1ⅹ106、2.5ⅹ105。与其它禽类病毒的交叉实验表明：这三株单抗具有良好的抗CIAV VP3蛋白特异性。该特异性单克隆抗体的研制成功为进一步研究VP3的生物学特性打下了基础。     

重组人瘦素蛋白纯化鉴定及其单克隆抗体制备

大肠杆菌（E.coli）重组表达获得的重组人瘦素蛋白（rh-leptin）,复性、纯化后进行SDS-PAGE电泳和Western-blot印迹杂交鉴定其免疫学活性,免疫小鼠后制备单克隆抗体,结果表明通过对rh-leptin进行复性和纯化,获得了高纯度的具有免疫学活性的rh-leptin蛋白,并获得一株稳定分泌抗rh-leptin单抗的杂交瘤细胞株。瘦素蛋白的纯化及其单克隆抗体的制备,可供瘦素进一步研究应用。     

Preparation of recombinant bovine, porcine, and porcine W4R/R5K leptins and comparison of their activity and immunoreactivity with ovine, chicken, and human leptins

Recombinant ovine Ala-leptin (GenBank Accession No. U84247, of ovine leptin), previously prepared in our laboratory in prokaryotic expression plasmid pMON3401, was mutated using a mutagenesis kit to prepare plasmids encoding for bovine (GenBank Accession No. U50365) and porcine (GenBank Accession No. U59894) leptins and for porcine leptin analogue W4R/R5K. Escherichia coli cells transformed with these plasmids overexpressed large amounts of these proteins upon induction with nalidixic acid. The expressed proteins, found in inclusion bodies, were refolded and purified to homogeneity using subsequently anion- and cation-exchange chromatography. All three purified proteins showed a single band of the expected molecular mass of 16 kDa in SDS-PAGE in the presence of reducing agent and were composed of 90-100% monomers. Proper refolding was evidenced by comparing their CD spectra to those of previously prepared chicken and ovine leptins and to commercially available human leptin. The amino acid content of the purified proteins closely resembled the predicted composition. The biological activity of bovine leptin, porcine leptin, and porcine leptin analogue W4R/R5K was evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor. All three proteins, as well as ovine and chicken leptins, but not human leptin, exhibited a very high degree of cross-immunoreactivity against antiserum raised against ovine leptin in rabbits. In contrast, none or very low cross-immunoreactivity was observed against antiserum raised against ovine leptin in goats.     

Products of cells from gliomas: VIII. Multiple-well immunoperoxidase assay of immunoreactivity of primary hybridoma supernatants with human glioma and brain tissue and cultured glioma cells

To test the feasibility of primary screening of hybridoma supernatants against human glioma tissue, over 5000 combinations of hybridoma supernatants with glioma tissue, cultured glioma cells, and normal central neural tissue were screened with a new multiple-well (M-well) screening system. This is an immunoperoxidase assay system with visual endpoints for screening 20-30 hybridoma supernatants per single microscope slide. There were extensive differences between specificities to tissue and to cultured glioma cells when both were screened with M-wells and when cultured cells were screened with standard semi-automated fluorescence. Primary M-well screening with glioma tissue detected seven hybridoma supernatants that specifically identified parenchymal cells of glioma tissue and that were not detected with cultured cells. Immunoreactivities of individual supernatants for vascular components (nine supernatants), necrosis (five supernatants), and nuclei (three supernatants) were detected. Other supernatants bound multiple sites on glioma tissue and/or subpopulations of neurons and glia of normal tissue. The results show that primary screening with glioma tissue detects a number of different specificities of hybridoma supernatants to gliomas not detected by conventional screening with cultured cells. These are potentially applicable to diagnosis and therapy.     

抗人内皮抑素单克隆抗体杂交瘤细胞的制备及筛选

用亲和层析纯化的酵母表达重组人内皮抑素为抗原,经皮下多点注射免疫Balb/c小鼠,鼠抗血清效价达到1：10000,选免疫效果最好的小鼠,取其脾细胞用PEG法与同系骨髓瘤细胞（SP2／0）融合,通过间接ELISA法对杂交瘤细胞进行筛选,对阳性孔进行有限稀稀,经3次亚克隆获得4株阳性杂交瘤细胞。     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

抗人血栓调节蛋白单克隆抗体的制备与鉴定

为了制备特异性抗人血栓调节蛋白(human thrombomodulin,hTM)的单克隆抗体(monoclonal antibody,McAb),利用脂质体Lipofectamine 2000将包含hTM全长cDNA序列的重组表达质粒pThr402转染CHO细胞,经G418筛选及相关鉴定后获得高效稳定表达hTM的CHO-TM5细胞株。将CHO-TM5细胞直接免疫Balb/c小鼠,应用杂交瘤技术,通过细胞ELISA (cellular enzyme-linked immunoabsorbent assay,CELISA)筛选出阳性克隆后,将杂交瘤细胞株腹腔注射Balb/c小鼠诱生腹水。用CELISA、流式细胞术、免疫组织化学染色法及免疫印迹法对所获McAb的特异性进行鉴定。我们获得了1株可稳定分泌抗hTM的McAb的杂交瘤细胞株NH-1,其亚型为IgGl,McAb腹水效价为1×10~(-6),腹水抗体含量为20 mg/ml。NH-1对相应抗原具有较高的组织特异性,在体内与正常组织的交叉反应少,对人脐静脉内皮细胞、CHO-TM5有特异性结合反应,说明NH-1可特异性识别天然的hTM分子,为进一步应用此McAb进行hTM生物学功能及临床意义研究提供了基础。     

Leptin inhibits steroid biosynthesis by human granulosa-lutein cells.

Absence of leptin secretion compromises reproductive function and fertility in the ob/ob mouse which, when given leptin, shows a rise in serum LH levels and becomes fertile. Recently, the long and active isoform of the leptin receptor was detected in the ovary, indicating that leptin may also show direct gonad-related activity. To examine this, we studied the effect of graded doses of human leptin on estradiol (E2) and progesterone (P4) concentrations in the culture media of human granulosa-lutein cells obtained from follicular fluid of women undergoing in vitro fertilization. We also evaluated the mRNA expression of steroidogenic acute regulatory protein (StAR), aromatase, and cytochrome P450 17alpha (CYP17) in these cells at baseline and after exposure to leptin. Estradiol levels were significantly decreased in the media 24 hours after incubation of the cells with increasing hLeptin concentrations (10(-11) - 10(-7) mol/l). The maximal 30% decrease in E2 production was caused by the 10(-9) mol/l hLeptin concentration; however, P4 levels in the media were not influenced by leptin. Exposure of granulosa-lutein cells to 10(-9) mol/l hLeptin did not produce any measurable changes on StAR, aromatase, or CYP17 mRNA expression. When hLeptin (10(-9) mol/l) was co-incubated with increasing concentrations of hCG (1.25 - 10 mlU/ml), IGF-II (15-60 ng/ml) or 1-6 desaminated IGF-II (deslGF-II; 15-60 ng/ml), it did not modify the elevation of E2 concentrations caused by each of the different stimuli. We conclude that leptin suppresses E2 secretion by human granulosa-lutein cells but does not impair the stimulatory effects of hCG and IGFs on these cells. Leptin may play a minor, but direct regulatory role on unstimulated human ovarian steroidogenesis by interfering with either the translational or post-translational steps of the baseline CYP17 and/or aromatase synthesis and/or the activation of the enzymes.     

Leptin modulates the expression of secreted and membrane-associated mucins in colonic epithelial cells by targeting PKC, PI3K, and MAPK pathways

Mucins play an essential role in the protection and repair of gastrointestinal mucosa. We recently showed that luminal leptin strongly stimulated mucin secretion in vivo in rat colon. In the present study, we challenged the hypothesis that leptin may act directly on goblet cells to induce mucin expression in rat and human intestinal mucin-producing cells (DHE and HT29-MTX). The endoluminal effect of leptin was also studied in vivo in rat perfused colon model. The presence of leptin receptors was demonstrated in the two cell lines by Western blot and RT-PCR. In rat DHE cells, leptin (0.01-10 nmol/l, 60 min) dose dependently increased the secretion of mucins (210 +/- 3% of controls) and the expression of Muc2, Muc3, and Muc4 (twofold basal level) but not of Muc1 and Muc5AC. Luminal perfusion of leptin (60 min, 0.1-100 nmol/l) in rat colon also increased the mRNA level of Muc2, Muc3, and Muc4 but not of Muc1. In human HT29-MTX cells, leptin (0.01-10 nmol/l, 60 min) dose dependently enhanced MUC2, MUC5AC, and MUC4 mRNA levels. These effects were prevented by pretreatment of cells with the leptin mutein L39A/D40A/F41A, which acts as a receptor antagonist. Finally, pathway inhibition experiments suggest that leptin increased mucin expression by activating PKC-, phosphatidyl inositol 3-kinase-, and MAPK-dependent pathways but not the JAK/STAT pathway. In conclusion, leptin may contribute significantly to membrane-associated and secreted mucin production via a direct stimulation of colonic epithelial cells and the activation of leptin receptors. These data are consistent with a role for leptin in regulation of the intestinal barrier function.     

Encapsulation of viable cells within polyacrylate membranes

A new, semipermeable hydrogel membrane for encapsulating viable cells has been developed. The encapsulation was performed by consecutively introducing droplets of a suspension of hybridoma cells in a solution of a polyanionic acrylic copolymer into aqueous solutions of three polycationic polymers. As a result of interpolymeric ionic interactions and some chemical reactions, a polyelectrolyte complex membrane was formed at the interface between each droplet and the polycationic polymeric solutions. The hybridoma cells, used as a model system, were derived by fusing spleen cells from immunized BALB/c mice with the NS-1 murine plasmacytoma cell line. The cells divided and gradually filled the microcapsules over a period of 8 days. Prior to encapsulation of the hybridoma cells, the polyanions were tested for toxicity and inhibition of cell growth. A direct relationship was observed between hybridoma cell viability in the acrylic polyanion/RPMI-1640/10% (w/v) fetal calf serum (FCS) solutions and the kinematic viscosities of the solutions of the polyanions. Antihuman IgM was produced by the encapsulated hybridoma cells and immunoassay showed that the antibody concentration was 3 mug/ml of the total culture medium.     

Human alveolar macrophage FcR-mediated cytotoxicity. Heteroantibody- versus conventional antibody-mediated target cell lysis

Human alveolar macrophage have three distinct receptors for IgG: FcRI, FcRII, and FcRIII. In order to compare the ability of these receptors to mediate target cell lysis, three different assay systems were examined. First, we studied lysis of chicken E (CE) opsonized with heteroantibodies, which are synthetic antibodies composed of Fab fragments with anti-FcR activity covalently linked to Fab fragments with anti-CE activity. We found alveolar macrophage readily lysed heteroantibody-opsonized CE via each of the three FcR classes (FcRI, 20 +/- 5%; FcRII, 27 +/- 7%; and FcRIII, 13 +/- 13%, p less than 0.05). Non-FcR-dependent lysis of anti-beta 2-microglobulin x anti-CE heteroantibody-opsonized CE was not detected. Second, lysis of hybridoma cell lines bearing anti-FcR antibodies on their cell surface was examined to assess killing of "tumor-like" target cells. Whereas peripheral blood monocytes and lymphocytes were able to lyse hybridoma cell lines bearing surface anti-FcR mAb, alveolar macrophages were not. Third, activity of alveolar macrophage FcR was examined in a conventional antibody-dependent cellular cytotoxicity assay by using O+ (R1,R2) human RBC opsonized with human anti-D and anti-CD serum as target cells. We found lysis of anti-D and anti-CD opsonized human RBC was mediated exclusively via FcRI. No activity of FcRII or FcRIII was detected in these latter assays even if performed under conditions that impair FcRI activity. Thus, all three FcR present on alveolar macrophage mediate lysis of heteroantibody-opsonized CE; in contrast, with the use of a conventional antibody-dependent cellular cytotoxicity assay, only FcRI activity was detected. We were unable to demonstrate lysis of anti-FcR-bearing hybridoma cell lines by alveolar macrophages.