COMPARISON OF FOUR METHODS TO GENERATE IMMUNOREACTIVE FRAGMENTS OF A MURINE MONOCLONAL ANTIBODY OC859 AGAINST HUMAN OVARIAN EPITHELIAL CANCER ANTIGEN

In the present study,four different proteases (pepsin,papain,bromelain and ficin) were screened with a murine monoclonal antibody OC859,in order to verify whether different digestion procedures could improve yield and stability of the F(ab‘)2 or Fab fragments.The yields of F(ab‘)2 or Fab fragments from digestion with pepsin,papain,bromelain and ficin were respectively 20.3 /-2.0%,50.5% /-5.0%,74.4 /-2.7% and 82.8 /-10.2% of the theoretical maximum.Immunoreactivity in a noncompetitive solid-phase radioimmunoassay (SPRIA) of the fragments generated by the four proteases were respectively 10 /-5%,36 /-5%,60 /-6% and 75 /-6% of the intact OC859 IgG.These results suggested that the fragmentation of OC859 with ficin gave a higher yield of superior immunoreactive fragments.     

PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

Objective.To generate phage-displayed anti-idiotypic single chain variable fragments(anti-Id ScFv) to MG7 monoclonal antibody(McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods.Balb/c mice were immunized i.p.with MG7 McAb conjugated with keyhole limpet hemocyain (KLH),and mRNA was isolated from the spleens of the immunized mice.Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR.The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1.The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage.After 4 founds of panning with MG7,the MG7-positive clones were selected by ELISA from the enriched phages.The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results.The VH,VL and ScFv DNAs were about 340 bp,320bp and 750 bp respectively.Twenty-four MG7-positive clones were selected from 60 enriched phage clones,among which 5 displayed β or γ type anti-Id ScFv.Conclusion.The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique,which paves a way for the study of prevention and cure of gastric carcainoma by using anti-Id ScFv.     

PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST HUMAN TELOME RASE REVERSE TRANSCRIPTASE

INTRODUCTIONTelomerasemaycontributetocellreplicationbymaintainingthelengthofcellulartelomeres.Normaltelomeraseactivityisrequiredtomaintainprolifera-tionofgermlineandstemcells,lackingofwhichisimportanttothesenescenceandagingofmostsomat-iccells.Reactivationoftelomeraseisessentialforcelltransformation.Telomeraseactivityhasbeende-tectedinmosthumancancers,whileinhibitionoftelomeraseactivityincancercellsleadstocancersuppression(1,2).Consequentlytelomerasemaybeanewdiagnosticmarkeroftumorsandtarg…     

THE DEVELOPMENT OF MONOCLONAL ANTIBODY AGAINST rhTNF AND ITS CURATIVE EFFECTS ON E. Coli INFECTED MICE

INTRODUCTIONTumornecrosisfactor(TNF),producedbymacrophagesandotherimmunocompetentcellsduringinfection,hasbeenidentifiedasapolypeptidecytokine(1)'TNFplayedanimportantroleinvariouspatho-physiologicalprocesses,andmediatedseveraldisorderswhenproducedinexcess(2).AhigherTNFlevelinpatientswithsepticshockusuallycausetissueinjury(1)-Recently,antagoniststoTNFa,suchasantibodyagainstTNFa(3~5),haveshowntobebeneficialinthetreatmentofbacteriainfection.Therefore,thereisaneedfordevelopmentofanti-TN…     

GENERATION AND PARTIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SOLUBILIZED MEMBRANE FRACTION OF HUMAN SPERMATOZOA

Human sperm membrane antigens extracted by deoxycholate (DOC) were used to immunizeBALB/c mice.Hybrid cell lines secreting sperm-specific monoclonal antibodies were generatedby cell fusion in a semi-solid medium and screened by indirect immunofluorescent assay usinglive and methanol-fixed sperm.Out of 850 hybrid clones from cell fusion,28 were shownto secrete sperm-specific antibodies which reacted with the acrosome,equatorial segment,whole surface plasma membrane or tail of spermatozoa.Finally,seven hybrid cell lineswere established and shown to secrete monoclonal antibodies which had no cross-reactivitywith arty human tissues other than testis and sperm.The majority were also shown toinhibit fertilization of mouse oocytes in vitro and human sperm penetration of zona-freehamster ova.Western blot analysis revealed that some of these antibodies reacted withsperm membrane antigens of distinct molecular size.     

抗CEA单克隆抗体对卵巢癌放射免疫显像研究

用~(131)I-抗CEA单克隆抗体对22例卵巢癌患者进行放射免疫显像研究,结果20例阳性显像,2例阴性显像(假阴性),20例阳性者结果与手术中所见相符,48处转移灶共发现45处,阳性率93.8％,最小检出病灶直径1.5cm,其中2例大网膜广泛转移和1例升结肠转移为采用本方法首次发现,显示放射免疫显像对卵巢癌不仅可以定性定位,还能检出转移灶,与B超、CT等方法比较具有一定优点,有较好的临床应用价值.     

抗卵巢上皮癌人单克隆抗体HMD_4的制备及其特性的研究

本文报道用人-鼠杂交技术制备出一株分泌人IgG型单克隆抗体的人-鼠杂交瘤细胞系,通过早克隆化和反复再克隆化等使该株细胞已稳定分泌15月以上。染色体核型分析符合人-鼠杂交瘤特征。免疫印迹和免疫组化等方法证实该抗体可识别分子量为55KDa的卵巢上皮癌相关抗原,命名为HMD_4。本文还对如何保持种间杂交瘤分泌功能和人单抗筛选中如何克服内源性Ig问题进行了讨论。     

抗肺癌单克隆抗体的研制

采用新鲜肺癌组织制成的细胞悬液及肺腺癌细胞株做为免疫原,免疫Balb/c小鼠。取脾细胞与经活体增殖的无支原体污染的高活力SP2/O骨髓瘤细胞融合,联合应用酶联免疫吸附和多组织切片免疫组化染色法进行筛选,得到两株分泌对肺癌具高特异性的单克隆抗体的杂交瘤细胞株。抗体与被检测的大多数人体正常组织无交叉反应,可初步用于肺癌病理鉴别诊断。     

PURIFICATION AND IDENTIFICATION OF THE ANTIGEN ASSOCIATED WITH MONOCLONAL ANTIBODY 3H11 AGAINST GASTRIC CANCER CELL

The object of this study is to investigate the properties of gastric cancer assoiciated antigen by McAb3H11 against gastric cancer cell line. Antigen purified by affinity chromatography and further characterized by biochemical and immunological techiques. Results showed that assoiciated antigen of McAb 3H11 was mainly located on the membrane of target calls. It was sensitive to heat and digestion of proteases,but resistant to treatment with sodium perioclate. Schiff‘s reagent staining was negative. SDS-PAGE and IEF-analysis showed that the antigen was a 210 kD molecular weight with pl of 8.5 protein. The molecular weight of antigen extracted from target cell with tunicamycin was not changed. We concluded that the corresponding antigen of MeAb 3H11 might be an alkaline protein.     

人肝癌培养细胞粗提膜相关抗原的单克隆抗体

用人肝癌培养细胞粗提膜免疫Balb/c小鼠的脾细胞与小鼠骨髓瘤(SP_2/o)细胞在PEG作用下融合。以LLISA筛选抗体。通过免疫荧光和免疫酶标进行组织定位。初步获得一株抗人肝癌细胞膜相关抗原的单克隆抗体杂交瘤—命名FP—5。用此抗体在涂片上对人肝癌细胞膜呈阳性反应,在3例手术切除的人肝癌活检冰冻切片中,肝癌组织细胞亦呈阳性反应。FP—5细胞株染色体为83—108条,含1—2条中着丝点标记染色体。经6次克隆化体外培养6个多月,仍持续分泌特异性抗体,此抗体鉴定属鼠的IgM型。     

抗人TPO单克隆抗体的制备

目的：制备抗人血小板生成素（hTPO）单克隆抗体，用于建立ELISA方法和检测TPO的表达。方法：用基因重组的hTPO免疫Balb／c小鼠，常规法做细胞融合，用ELISA法筛选出阳性杂交瘤细胞株。结果：获得一杂交瘤细胞株（46-6），其免疫球蛋白亚类为IgG1经ELISA和免疫印迹技术证明，此单抗能特异识别TPO。结论：46－6单克隆抗体（McAb）特异性强。用其建立的夹心ELISA检测TPO的方法线性好，灵敏度可达50ng／L。     

抗胃癌及丝裂霉素C双功能抗体的制备

用杂交瘤与脾细胞融合的方法制备了可分泌抗胃癌及丝裂霉素C(MMC)双功能单克隆抗体的二次杂交瘤(hybrid-hybridoma),该双功能抗体由γ_(2b)链和μ链构成,游离MMC对其与HSA-MMC结合的50％抑制浓度为1.46μg/ml。     

单克隆抗体免疫亲和层析一步法纯化癌胚抗原

本文用特异性抗癌胚抗原的单克隆抗体,经与CNBr—Sepha?ose4B偶联,以100mmol/L二乙胺pH11.5洗脱亲和层析柱,一步法从NP—40可溶性的大肠腺癌组织提取物中纯化癌胚抗原。以SDS-聚丙烯酰胺凝胶电泳和Western-Blotting鉴定了抗原纯度及活性。以微量固相免疫放射试验检测了抗原活性。     

抗人胰腺癌单克隆抗体杂交瘤的建立

用小鼠骨髓瘤细胞系SP 2/0与新鲜癌组织匀浆免疫的Balb/C小鼠脾细胞融合制备抗人胰腺癌单克隆抗体。经间接荧光抗体法和免疫组织化学法筛选及有限稀释法克隆化后,获得一株产生与正常人体组织不反应,与人胰腺癌反应率高的抗人胰腺癌单克隆抗体的杂交瘤细胞株。