抗狂犬病病毒CVS-11株单克隆抗体的制备及鉴定

目的 制备抗狂犬病毒单克隆抗体,为建立快速准确的狂犬病毒抗原检测方法奠定基础.方法 将狂犬病病毒CVS-11株纯化浓缩后免疫6～8周龄雌性BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经细胞克隆和间接ELISA筛选,获得稳定分泌抗狂犬病毒单克隆抗体的杂交瘤细胞株.小鼠腹腔注射法制备大量单克隆抗体并测定腹水效价,用G蛋白亲和层析柱进行纯化,间接ELISA法和间接荧光法鉴定单克隆抗体的类型、特异性及敏感性.结果 细胞融合率达100%,经克隆筛选获4株稳定分泌抗狂犬病病毒抗体的杂交瘤细胞株,其腹水效价分别为1×104,1×105,1×104和1×105;4株单抗均为IgG类型且特异性好.结论 制备的单抗具有良好特异性和敏感性.     

抗人BPI23单克隆抗体的制备和鉴定

目的 采用杂交瘤技术制备抗人BPI23单克隆抗体,并对其应用进行初步分析。方法 免疫小鼠脾细胞与小鼠骨髓瘤细胞按常规方法融合;用间接ELISA法和Western - blot筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法亚克隆3次获得稳定分泌抗人BPI23单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注入小鼠腹腔后制备腹水;纯化腹水中的单抗并对抗体类型进行鉴定;用Western-blot分析抗体的特异性;用间接ELISA法测抗体效价;将分离纯化的正常人外周血中性粒细胞和单个核细胞制成涂片,用抗人BPI23单克隆抗体进行免疫染色。结果 获得3个（1B4、9C12和2H11）稳定分泌抗人BPI23单克隆抗体的杂交瘤细胞株,所分泌的单抗类型分别为κ型IgM、κ型IgG1和κ型IgG1;抗体效价分别为1.28×105、1.28×105和4.1×106,纯化后抗体含量分别为0.208g/L、2.03g/L和3.88g/L;3种纯化抗体均能与本实验制备的人BPI23和市售人BPI55标准品特异性结合,而不能与小鼠BPI25和人LBP结合;在免疫组化实验中,1B4、9C12和2H11单抗均能与人中性粒细胞中的BPI特异性结合。结论 成功制备了人BPI23特异性单克隆抗体,为BPI检测试剂盒的研制奠定了基础。     

抗人类免疫缺陷病毒gp41和丙型肝为病毒NS3杂交？…

目的 为检测血清中人类免疫缺陷病毒和丙型肝炎病毒抗原提供血清学指标。方法 用纯化的重组ＨＩＶ和ＨＣＶ抗原蛋白作为联合免疫原,免疫ＢＡＬＢ／ｃ小鼠,取脾细胞与Ｓ２／０小鼠骨髓瘤细胞融合,并采用挑单个细胞的方法进行一次克隆。结果 分别获得４株和６株能稳定分泌高效价抗ＨＩＶ和ＨＣＶ抗的蛋白单克隆抗体杂交瘤细胞株。     

抗人类免疫缺陷病毒gp41和丙型肝炎病毒NS3杂交瘤细胞株的建立

目的为检测血清中人类免疫缺陷病毒（ＨＩＶ）和丙型肝炎病毒（ＨＣＶ）抗原提供血清学指标。方法用纯化的重组ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原蛋白作为联合免疫原，免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与Ｓｐ２／０小鼠骨髓瘤细胞融合，并采用挑单个细胞的方法进行一次克隆。结果分别获得４株和６株能稳定分泌高效价抗ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原蛋白单克隆抗体杂交瘤细胞株。初步建立了双抗体夹心法检测ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原的酶联免疫吸附试验。结论本方法是建立单抗杂交瘤细胞株的快速方法，所建杂交瘤细胞株特异性强，效价高，分泌抗体性能稳定，有推广价值。     

抗人红细胞M型抗原单克隆抗体的研制及初步应用

应用杂交瘤枝术,用人M型RBC免疫的BALB/c小鼠脾细胞,与小鼠骨髓瘤细胞系NS-1融合,筛选出抗M血型抗原的单克隆抗体杂交瘤细胞株13H_2;经过8个多月连续传代培养增殖良好,仍能稳定分泌效价高、特异性强的单克隆抗体。细胞培养上清效价1∶512,亲和力11s。通过将此株单抗用于血型检测、标准红细胞谱细胞的鉴定和在法医上检测血痕,均证明此株单抗识别的只是红细胞上的M血型抗原,与其它血型抗原无关。较多克隆抗M型抗原血清特异性强,效价高,亲和力好,是检测MN血型的良好诊断试剂。     

抗早孕因子单克隆抗体的制备与鉴定

目的建立分泌抗EPF(Early pregnancy factor,早孕因子)单克隆抗体的杂交瘤细胞株,纯化单抗并鉴定.方法用本实验室已纯化的早孕和肿瘤源性EPF作为抗原刺激Balb/c小鼠,用免疫后的小鼠脾细胞与同系小鼠骨髓瘤细胞(NS-1)融合,经4次克隆化,获得可稳定分泌抗EPF单克隆抗体的细胞株,注入Balb/c小鼠腹腔制备腹水型单抗,Protein-A亲和层析纯化,SDS电泳和Western-blot等方法分析纯化结果.结果融合后获得一株稳定分泌抗EPF抗体的细胞株(C3D11),克隆化后,获得稳定分泌抗EPF单克隆抗体的细胞株,将增殖后的细胞注射Balb/c小鼠腹腔获得腹水型单抗,以亲和层析法纯化,SDS-PAGE分析显示纯化后去掉了大部分杂蛋白,免疫印迹分析抗体纯度较高,与抗原匹配性良好.结论本研究制备的EPF单克隆抗体为特异性抗EPF抗体.     

抗牛安氏隐孢子虫单克隆抗体的研制与初步鉴定

通过流行病学调查分离到奶牛隐孢子虫虫株,经实验室鉴定为牛安氏隐孢子虫(Cryptosporidiumandersoni),经蔗糖漂浮法收集和蔗糖密度梯度离心、滤器滤过、多次低速离心提纯后,通过冻融和超声波处理的全卵囊抗原免疫Balb/c小鼠4次,取阳性小鼠脾细胞与SP2/0骨髓瘤细胞融合。经间接ELISA方法筛选,有限稀释法3~4次克隆,获得5株分泌抗牛安氏隐孢子虫(C.andersoni)单克隆抗体的杂交瘤细胞株(1A3,3B10,3F4,4B3,4F11)。5株单抗的染色体数目平均在96~98条之间,上清效价分别在1∶100和1∶12800之间,腹水效价分别在1∶12800和1∶204800之间。采用免疫荧光法对各株的单抗腹水进行初步鉴定,发现其中4株为抗卵囊壁单抗,1株为抗子孢子单抗。对其中3株单抗的腹水提纯后进行SDS-PAGE电泳分析,其重链分子量约为52·7kDa,轻链分子量约为24kDa。     

抗SARS病毒N蛋白单克隆抗体的制备和初步应用

目的 制备抗SARS病毒N蛋白的单克隆抗体并研究其初步应用。方法 用基因重组N蛋白免疫小鼠，取免疫后的鼠脾细胞与骨髓瘤细胞融合，筛选分泌抗SAPS病毒N蛋白单克隆抗体细胞株。将阳性细胞株接种小鼠腹腔制备单克隆抗体腹水并对抗体进行纯化，分析纯化抗体的相对亲和力。选择亲和力较高的抗体制备检测SARS病毒抗原的酶联免疫诊断试剂，并对其敏感性和特异性进行分析。结果 共获得11株单克隆抗体细胞株，其中3株单抗与N蛋白具有较高的亲和力，4株纯化单抗与N蛋白反应很弱，其余4株单抗介于两者之间。用亲和力较高的单抗制备检测SARS病毒抗原的诊断试剂，其敏感性可达31PFU／ml，而且与其他呼吸道病毒无交叉反应。结论 该试剂特异性较好，可用于SARS病毒抗原的检测，其敏感性仍需用临床急性期样品进行评价。     

平榛主要过敏原Corh1单克隆抗体的制备与鉴定

目的制备和鉴定鼠抗平榛主要变应原Cor h 1的单克隆抗体(Monoclonal Antibody,McAb)。方法用重组Cor h 1蛋白为免疫原,免疫Balb/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。通过间接ELISA法筛选分泌特异性McAb的杂交瘤细胞。用杂交瘤细胞株诱导小鼠产生腹水,再用蛋白A亲和层析法纯化抗体。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA、Western Blotting鉴定该McAbs的特性和交叉性。结果获得4株可稳定分泌鼠抗平榛主要变应原Cor h 1的McAbs,其Ig亚型均为IgG1,均具有良好的效价;ELISA和Western Blotting分析表明该4株单抗均能识别重组Cor h 1蛋白,其中3株单抗能够识别天然平榛提取物。结论成功制备了4株鼠抗平榛主要变应原Cor h 1的单克隆抗体,为建立平榛主要变应原Cor h 1检测及纯化方法奠定了基础。     

抗早孕因子单克隆抗体的制备与鉴定

目的建立分泌抗EPF(Early pregnancy factor，早孕因子)单克隆抗体的杂交瘤细胞株，纯化单抗并鉴定。方法用本实验室已纯化的早孕和肿瘤源性：EPF作为抗原刺激Balb／c小鼠，用免疫后的小鼠脾细胞与同系小鼠骨髓瘤细胞(NS-1)融合，经4次克隆化，获得可稳定分泌抗EPF单克隆抗体的细胞株，注入Balb／c小鼠腹腔制备腹水型单抗，Proteirr-A亲和层析纯化，SDS电泳和Western-blot等方法分析纯化结果。结果融合后获得一株稳定分泌抗EPF抗体的细胞株(C3D11)，克隆化后，获得稳定分泌抗EPF单克隆抗体的细胞株，将增殖后的细胞注射Balb／c小鼠腹腔获得腹水型单抗，以亲和层析法纯化，SDS-PAGE分析显示纯化后去掉了大部分杂蛋白，免疫印迹分析抗体纯度较高，与抗原匹配性良好。结论本研究制备的EPF单克隆抗体为特异性抗EPF抗体。     

Age-related natural antibody specificities among hybridoma clones originating from NZB spleen.

In the studies presented here, age-related natural antibody specificities have been investigated in the autoimmune NZB mouse strain by cell fusion. The monoclonal immunoglobulins (MIg) secreted by productive hybridoma clones were examined for their antibody activities against a panel of antigens, including single- and double-stranded DNA, actin, tubulin, myosin, bromelain-treated mouse red blood cells (BrMRBC) and TNP-BSA, employing both direct and competitive enzyme immunoassays. The antibody specificities against this panel of antigens were strikingly frequent among hybridoma clones from neonatal NZB (49%) mice, compared to normal BALB/c neonates (8.8%) shown earlier. Among neonatal hybridomas with known antigen reactivities, 73% of the clones exhibited polyspecific binding. In contrast, the majority of hybridomas from 5- and 7-month-old NZB spleen reacted monospecifically (76%) with the antigens tested. Such a characteristic reactivity pattern reflects an age-related evolution of B-cell repertoire expression. Unlike normal BALB/c mice, a high frequency of monospecific TNP-hapten-reactive clones (75%) was noticed among hybridomas of known antigen reactivities from 5- and 7-month-old NZB-strain mice, an age when autoimmune haemolytic anaemia sets in. In conclusion, an elevated frequency of autoreactive clones among neonates (49%) and an aberrant expression of TNP-reactive clones in adults seem to be an outward signal of certain discrepancies at the level of B-cell repertoire expression in autoimmune NZB-strain mice.     

Monoclonal antibodies to human cytomegalovirus: three surface membrane proteins with unique immunological and electrophoretic properties specify cross-reactive determinants

Seventy-seven clones of hybridomas selected for reactivity by immunofluorescence with human cytomegalovirus (CMV)-infected cells were produced by fusing mouse myeloma cells with the spleen cells of mice immunized with CMV strain AD169. The clones were classified into seven groups on the basis of the electrophoretic properties of the polypeptides immune precipitated from extracts of CMV-infected cells. Studies on the three groups of monoclonal antibodies directed against CMV surface membrane antigens showed the following. Clones in each group were differentiated by immunoglobulin subclass, neutralizing activity, and reactivity with the antigenic domains of proteins exposed on the surface membranes of intact CMV-infected cells. Monoclonal antibodies in each group precipitated one slowly migrating protein and multiple faster migrating forms which shared antigenic determinants. The first group of monoclonal antibodies precipitated four glycosylated polypeptides with apparent molecular weights of 130,000, 110,000, 100,000, and 60,000. Monoclonal antibody CH51 of this group neutralized infectious virus but failed to react with antigenic domains on the surfaces of infected cells. The second group of monoclonal antibodies precipitated four polypeptides with apparent molecular weights of approximately 66,000, 55,000, 50,000, and 46,000. Monoclonal antibodies CH65 and CH134 in this group had neutralizing activity and reacted with antigenic domains of proteins exposed on the surface of CMV-infected cells. The third group of monoclonal antibodies precipitated four polypeptides with apparent molecular weights of 49,000, 48,000, 34,000, and 25,000. Serological analysis of 15 naturally occurring CMV strains with a panel of monoclonal antibodies to surface membrane proteins showed that the antigenic determinants reactive with the antibodies tested were conserved in all of the strains. Monoclonal antibodies to surface membrane proteins on CMV-infected cells may prove to be valuable reagents for identification of virus isolates.     

Rapid production of monoclonal antibodies to T lymphocyte functional antigens

Brief immunization of rats with mouse lymphoid cells was combined with the rat/mouse hybridoma technology and functional hybridoma screening to yield a rapid method for the production of monoclonal antibodies (MAb) against functionally important T lymphocyte cell surface antigens. Two protocols were used. In one, rats were immunized once with mouse thymocytes followed by fusion and screening of the hybridomas for interference with the thymocyte co-stimulator (interleukin 1) assay. The resultant hybridomas included producers of MAbs against the L3T4-antigen (inhibitory), the Ly-1-antigen (stimulatory), and the Thy-1-antigen (inhibitory?). In the second protocol, rats were immunized twice with a T cell hybridoma. The resultant hybridomas were screened for inhibition of polyclonal T cell activation, induced by an anti-Thy-1 (MAb G7). A panel of MAbs against the Thy-1 antigen with different reactivity profiles was generated by this procedure. Most of the MAbs were of the IgM class. Short-term immunization may lead to less selection of response to highly immunogenic determinants than a protocol involving several boosters. Thus, this alternative may be useful for producing MAbs against rare or weakly immunogenic cell surface molecules, as suggested by the ease with which we were able to make MAbs against the L3T4-molecule.     

Application of monoclonal antibodies in the avian leukosis virus GS-antigen ELISA

Five hybridoma cell lines which secrete antibodies to avian leukosis/ sarcoma (ALV) group-specific (gs) antigens (gag gene products) have been established. The hybrid cells resulted from fusion of P3/X63-Ag8.653 myeloma cells with splenocytes of BALB/c mice which had been immunised with purified avian myeloblastosis virus (AMV). Screening of supernatant fluids was performed by an indirect double antibody sandwich enzyme-linked immunosorbent assay (IDAS-ELISA) and the immunoelectroblotting technique. Three hybrid clones secrete monoclonal antibodies (MCA) to 27,000 dalton polypeptides (p27) and two hybridomas produce monoclonal antibody directed to 19,000 dalton phosphoprotein (pp19). All five monoclonal antibodies belong to mouse immunoglobulin isotype IgG(1). MCAs directed to different ALV-p27 epitopes can replace polyclonal rabbit or hamster anti-gs sera in the DAS-ELISA in use for the detection of congenitally ALV-shedding hens. In albumen samples a 16- to 32-fold increase of sensitivity of ALV gs-antigen detection was obtained as compared to the DAS-ELISA employing polyclonal sera. Gs-antigens of a purified AMV preparation were detectable up to minimal concentrations of 13 pg/ml.     

抗人4-1BB功能性单克隆抗体的研制及生物学特性研究

研制鼠抗人4-1BB功能性单克隆抗体及其在T细胞活化、增殖及信号转导中的作用。以小鼠肾肿瘤细胞转染人4- 1BB基因的细胞株4-1BB/293T为免疫原.采用B淋巴细胞杂交瘤技术,获取分泌特异性4-1BB单抗的杂交瘤细胞株。以体内诱生法产生腹水,Protein G亲和层析法进行纯化,快速定性试纸法鉴定单抗的亚类,MTT法检测单抗对T细胞的刺激作用,流式法检测单抗对T细胞凋亡的影响。结果显示:成功获得1株持续分泌特异性抗人4-1BB单抗的细胞株(命名为5D5).属于IgG2b。该单抗能特异识别人4-1BB分子,介导有效的共刺激信号,体外促进T细胞的增殖,抑制活化诱导的T细胞凋亡。总之,成功获得1株功能性4-1BB单抗,具有重要的研究和潜在应用价值。     

Generation and characteristics of hybridomas producing monoclonal antibodies to the surface antigen of hepatitis B virus

An optimal schedule for immunization of BALB/c mice has been found, providing a high yield of hybridoma clones producing monoclonal antibody (MCA) to hepatitis B virus surface antigen (HBsAg). Fourteen hybridomas of ZHAK series have been prepared. The cells of 9 hybridomas secrete MCA to the common-type determinant a, and of 5 hybridomas, to the subtype determinant y of HBsAg. The capacity of hybridoma cells for clone production and for induction of ascitic tumors in syngeneic animals was studied. In the cells of two clones marker chromosomes were detected not occurring in the original parent cells.     

Serological analysis of dermatophyte isolates with monoclonal antibodies produced against Microsporum canis.

Hybridoma cells produced by fusing myeloma cells with spleen cells from mice immunized with a soluble antigen of Microsporum canis yielded 30 antibody-producing clones. Six of these clones, propagated as ascites tumors in mice, showed two different types of monoclonal antibodies. The type 1 monoclonal antibody reacted with 17 heterologous and 10 homologous dermatophyte antigens. Type 2 monoclonal antibodies were unable to precipitate three antigens from different isolates of M. canis, thus suggesting the occurrence of different serotypes within the species.     

抗HIV-1核心抗原p24单克隆抗体的制备及特性的初步鉴定

目的 :建立分泌抗HIV 1p2 4单克隆抗体 (mAb)的杂交瘤细胞株 ,并对其特性进行初步鉴定。方法 :以纯化的基因工程制备的p2 4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HAT、HT选择培养及有限稀释法进行克隆化后 ,用间接ELISA法及Dotblot对其进行筛选和特性鉴定。结果 :筛选到 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,其腹水效价为 1×10 -5,亲和力为 1.7× 10 4～ 1.8×10 4mol/L ,mAb的Ig亚类均为IgG1。两株mAb与HBcAg、HCVRNA阳性血清及HIVgp4 1等均无交叉反应 ,只与HIV 1p2 4抗原阳性血清产生特异反应。结论 :成功地建立了 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,为进一步研制HIV 1p2 4抗原的ELISA检测试剂盒奠定了基础