A panel of monoclonal antibodies against the prion protein proves that there is no prion protein in human pancreatic ductal epithelial cells

Prion diseases are a group of neurodegenerative diseases that are fatal. The study of these unique diseases in China is hampered by a lack of resources. Amongst the most important resources for biological study are monoclonal antibodies. Here, we characterize a panel of monoclonal antibodies specific for cellular prion protein by enzyme-linked immunosorbent assay(ELISA), immunofluorescent staining, flow cytometry, and western blotting. We identify several antibodies that can be used for specific applications and we demonstrate that there is no prion protein expression in human pancreatic ductal epithelial cells(HPDC).

MONOCLONAL ANTIBODIES AGAINST HUMAN GASTRIC CANCER

Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) against gastric cancer, were obtained through selective culture and screening. These MoAbs have both good selectivity and a high positive rate of reaction for gastric cancer, reaching 5/5 and 84.8% to 93.5% for gastric cancer cells and tissues respectively. The reaction of MoAbs with normal cells and tissues was neglible.The corresponding antigens of the MoAbs were sensitive to digestion by trypsin and pronase and resistant to treatment with sodium periodate, indicating their nature as proteins. The antigen 3G9 could be visualized with Western blotting as two bands with molecular weights of 100KD and 70KD, however no band was found for antigens 3F4 and 3H11. There was a high expression of antigens in the majority of gastric cancer cells and tissues independent of his-topathological type of gastric cancer. A low expression of antigens was seen with other tumors and fetal gastrointestinal tissues. These could be considered as gastric cancer-associated antigens or on-cofetal antigens with a quite extensive distribution.

Enhanced radioimmunotherapeutic efficacy of a monoclonal antibody cocktail against SMMC-7721 human hepatocellular carcinoma

The improved tumoricidal effect of the radioatibody mixture ("cocktail") has been reported recently for the treatment of colon tumor. In the present study, we demonstrated the enhanced radioimmunotherapeutic efficacy of a monoclonal atibody (MAb) cocktail against human hepatocellular carcinoma. Therapeutic efficacy was determined by measuring the change in tumor size over a period, determining the percentage of growth inhibition of each treatment at various times after radioantibody therapy. boioimmunotherapy of SMMC-7721 human hepatoma xenografts in athymic nude mice with combination of 131I labeled Hepama-1 and 131Llabeled 9403 mouse MAbs was more effective than using either Hepeam-1 or 9403 Mab alone The MAb cocktail could target a greater number of hepstoma cells and increase the magnitude of hepatoma cen uptde of radioamibodies. The in vjtro results explain the enhanced effect of the MAb cocktail in in vjvo model system.

PURIFICATION OF RECOMBINANT HUMAN INTERFERON-γ BY IMMUNOAFFINITY CHROMATOGRAPHY WITH MONOCLONAL ANTIBODY

E.coli cells expressing recombinant human interferon-γ was disrupted by sonication anddissolved in 7mol·L-1 guanidine hydrochloride.The extract obtained was then renaturated by 70 folddilution with PBS.HulFN γ was purified by affinity chromatography with monoclonal antibody fromthe renaturated crude feed solution.After washing the column with PBS,the adsorbed HulFN γ waseluted with PBS containing 0.5mol·L-1 NaCl.The column was regenerated with 2mol·L-1 GuHClfor reuse.After one step of affinity purification the purity of interferon-γ was over 95%.and thespecific activity of the HulFN-γ reached 1.2×107 IU·mg-1 protein.92.8% of recovery was obtainedin the elution step.Total recovery of HulFN γ activity in the affinity chromatography was 78%.

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.

Production and Characterization of Monoclonal Antibody Against Recombinant Human Erythropoietin

Objective To produce specific monoclonal antibody （mAb） against recombinant human erythropoietin （rHuEPO） for development of highly efficient methods for erythropoietin detection in biological fluids. Methods rHuEPO was covalently coupled with bovine serum albumin （BSA） and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked irmnunosorbent assay （ELISA）, SDS-PAGE and Western blot. Results The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1x10s L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work. Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.

THE STUDIES ON MONOCLONAL ANTIBODY SZ-39 AGAINST HUMAN GLIOMA CELLS

A hybridoma cell line SZ-39 secreting monoclonal antibody against the human glioma cell has been established by a fusion between NS-1 myeloma cells and spleen cells from mice immunized with human glioma cell lines. Monoclonal antibody (McAb) SZ-39 was analyzed by ELISA, quantitative absorption, indirect immunofluorescence and ABC immunohistology. McAb SZ-39 strongly bound to 9/10 glioma cell lines, 17/20 glioma tissues, weakly bound to one liver cancer cell line and 1/2 lung cancer line, but they did not band with other tested human cancer linse. NcAb SZ-39 have no cross-reaction with lymphocyte, ABC red blood cells, white blood cells, blood platelet, normal bone marrow cells, fibroblast cells and 12 normal human tissues.The result indicated the antigen recognized by McAb SZ-39 may be a glioma-associated antigen <GAA). This GAA was analyzed by means of Western blotting. It was a MW 180 Kd glycopro-tein. The 131I-McAb SZ-39 specifically localized in human glioma xenografted in nude mice that indicate it may be useful in radioimmunoimaging and as a target for immunotherapy on human glioma.

SYNTHESIS OF INTERFERON-α_A MONOCLONAL ANTIBODY PACKING MATERIAL IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PURIFICATION OF RECOMBINANT HUMAN INTERFERON-α_A

A new way for the synthesis of human interferon—α;monoclonal antibody （IFN-α;-McAb） bound to silica gel packing material in high-performance affinity chromatography （HPAFC） has been developed. The high coupling efficiency and specific activity of IFN—α;-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α;（rIFN-α;） rose up to 1.03×10;IU/mg protein and the purification efficiency is appoximately 100 times.

THE ALTERNATIVE PATHWAY OF HUMAN T CELL ACTIVATION BY MONOCLONAL ANTIBODIES(A COMPARATIVE STUDY BETWEEN NORMAL INDIVIDUALS AND CANCER PATIENTS)

This paper described T cell proliferative response by an alternative pathway in normal subjects and In patients with malignant diseases. Two McAbs, Anti-CCTl and Lo-CD2-act recognizing two distinct epitopes on E-receptor (CD2) were used to costimulate PBMC. Proliferative responsiveness was measured by 3H-thymidine incorporation. It was found that 82% of 72 nonnal subjects gave proliferative response whereas only 23% of the 93 patients did. The average cpm±SD in patients with bladder cancer (118±2314), kidney cancer (1619±2719) or lymphoma (2518±4057) was significantly lower than that in normal subjects (4935±2314), (P<0.001). These results indicate that T cell proliferation through the alternative pathway was significantly depressed in patients with cancer, and this can be used as a new parameter to monitor the immune status of cancer patients.

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

Objective.To develop monoclonal antibodies again st the catalytic subunit of human telomerase reverse transcriptasefor i ts expression detection of human tumors.Methods.A dominant epitope in hTERT was automatically synthesized based on Fmoc method,and was used to immuni ze Balb/c mice.Hybridomas were generated and screened by ELISA for specific mo noclonal antibodies,and the characterization was performed by Western blotting and immunohistochemical staining.The heavy chain variable region of antibody wa s cloned by RT-PCR and sequenced.Results.Antigenic peptide hTERT 7 was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis.One hybr idoma cell line secreting anti-hTERT 7 antibodies designated as M2was established after primary screening and cons equent 3rounds of limited dilution.M2was IgG1in isotyping.The competi-tive assay showed that the M2antibody was hTERT 7-specific,and the affinity constant was about 1×10 6 mol-1 .The antibody reacted with cell extracts from HeLa cancer cells but not wi th those from normal2BS cells in ELISA assay.For in situ staining of immunohis tochemistry,the positive staining presented in the nuclear compartment of HeLa ,while2BS was negative.The heavy chain variable region from M2re-vealed tha t the monoclonal antibody was mouse origin.Conclusions.The developed mouse mon oclonal antibody is hTERT-specific and able to recognize native cellular hTERT in ELISA and immunohistochemistry,which makes the immuno-detection of telom-e rase hTERT expression in cancer cells or tissues possible.

HISTOLOGICAL RECOGNITION SPECTRUMS OF 4 MONOCLONAL ANTIBODIES AGAINST CYTOKERATINS

The histological specificities of 4 monoclonal antibodies against cytokeratins (HK2, HK5, K174 and K27) were investigated in various kinds of human and rat tissues with ABC immunohistochemical technique. K174 was shown to have the same recognition spectrum with a polyclonal antibody (RAK1) to epidermal keratin. HK2 and HK5 were similar except the difference in reaction with stratified squqmous epithelium. K27 could only label the suprabasal layers of the squamous epithelium, and could be used as a marker of cornified or cornifying epithelium. This study provided a sound basis for the use of this group of antibodies in the subtyping of different epithelial tissues and the tumors originating from them.

Establishment and application of immunofluorescence using monoclonal antibody in diagnosis of gastric cancer

Monoclonal antibodies against gastric cancer(MG)were purified by ion-exchangechromatography on DEAE-cellulose(DE-52)column and coupled with CNBr-activated Sepharose4B.The Sepharose 4B linked with MG monoclonal antibodies was then incubated with serum andascitic fluid of patients with gastric cancer and benign diseases for 2h.After being blocked by nor-mal mouse serum,the MG-Sepharose was mack to react with the MG-labeled with fluoresceinisothiocyanate for 30min.According to immunoaffinity chromatography principle,the MG-labeledwith fluorescein isothiocyanate was separated from the MG-Sepharose with 0.01 mol/L of PBS-3mol/L of KCNS solution and then detected with a fluorospectrometer.The amount of fluorescencein the solution of separation represented the amount of the antigens defined by MG monoclonalantibodies.The mean value plus 2 standard deviations of the benign disease group was arbitrarilyset as their positive threshold.The positivity rates were 61.1%(22/36)in serum and 75%(18/24)inascitic fluid of patients with gastric cancer.It suggests that the determination of MG-Ags in the se-rum and ascitic fluid of patients may be helpful for the diagnosis of gastric cancer.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNAs encoding variable domains ofheavy and light chains(VH and VL) oftthe antibody wereamplified separately byRT-PCR and assembled into ScFvDNA with a linker DNAThe ScFv DNA was iigated into thephagemid vector pCANTAB5E and the ligated sample wastransformed into E. coil TG1. The transformed cells wereinfected with M13KO7 helper phage to yield recombinantphages. After two rounds of panning with gastric carcinomacell line AGS highly expressing MC3-binding antigen, thephage clones displaying ScFv fragments of the antibodywere selected by ELISA. 4 phage clones showing strongsignal in ELISA were used to infect E. coil HB2151 toexpress soluble ScFvs. The soluble ScFve were identified byDot blot and Western blot, and their antigen-binding activitywas assayed by ELISA. The VH and VL DNAs of the ScFvDNA derived from phage clone 19 were sequenced.RESULTS: The VH, VL and ScFv DNAs were about 340 bp,320 bp and 750 bp respectively. After two rounds of panningto the recombinant phages, 18 antigen-positive phageclones were selected from 30 preselected phage clones byELISA. All the soluble ScFvs derived from the 4 out of the 18antigen-positive phage clones were about Mr 32 000 andconcentrated in periplasmatic space under the given culturecondition. The soluble ScFvs could bind the antigen, andthey shared the same binding site with MC3. The sequencesof the VH and VL DNAs of the MC3 ScFv showed that thevariable antibody genes belonged to the IgG1 subgroup,κ-type.CONCLUSION: The soluble ScFv of MC3 is successfullyproduced, which not only provides a possible novel targetingvehicle for in vivo and in vitro study on associated cancers,but also offers the anuibody a stable genetic source.

Preparation, characterization and radiolabelling of antigranulocyte monoclonal antibody

The human granulocytes were isolated.Using hybridoma techniques,a hybridoma cell line (HSN) producing monoclonal antibody(McAb) against human granulocyte was obtained.The antibody belonged to IgG1 subclass.It was confirmed by immunohistochemical tests that HSN reacted selectively not only with human granulocytes.but also with their bone marrow precursors.Whereas human lymphocytes and red blood cells retained negative in the tests.No cross-reaction was observed with the peripheral blood cells in other animals.Its affinity constant was 5.7×10^8 L/mol,and the number of epitopes per granulocyte was 4.7×10^5.Monoclonal antibody displayed no loss of immunoreactivty after labelled with ^99mTc.

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

Fifteen hybridoma cell lines producing monoclonal antiboclies (McAb) against recombinant human tu-mor necrosis factor a (rhTNFa) have been established by fusing SP 2/0 cells with spleen cells from aBALB/c mouse immunized with rhTNFa. Following J M Davis’s Works, semi-solid medium was usedfor initial cloning. Five of them were studied further. Their main chromosome- numbers range were 96 to105, all of them were IgG1 subclass. The affinities of these McAbs were estimated to be 1. 25 ×108 mol/L, 1. 12×108 mol/L, 2. 34×108 mol/L, 8. 55 × 107 mol/L, 1. 04×108 mol/L, respectively.Two groups of mice challenging with E Coli (107 organisms), one group treated with 2mg/kg anti-TNF monoclonal antibody, the other did not. There was a higher survival rate in treated group, the serumTNF level was significantly lower too, and the untreated mice had severe pathologic changes in vlscera.

靶向HER2胞外结构域Ⅳ的单克隆抗体在乳腺癌中的应用进展

人表皮生长因子受体2(HER2)阳性乳腺癌侵袭性强且易转移,抗HER2靶向药物的应用能显著改善HER2阳性乳腺癌患者的预后。在已上市的HER2靶向药物中,靶向HER2胞外结构域Ⅳ的大分子单克隆抗体是治疗HER2阳性乳腺癌的基础靶向药物,主要包括曲妥珠单抗、伊尼妥单抗和马吉妥昔单抗。曲妥珠单抗用于乳腺癌全线治疗,循证医学证据充分,实践经验充足且安全性可控;伊尼妥单抗与曲妥珠单抗在HER2阳性转移性乳腺癌和新辅助/辅助治疗中疗效相似,且安全性可控;马吉妥昔单抗聚焦于携带CD16A-158F等位基因的患者,是晚期乳腺癌后线治疗的选择。临床上需根据患者具体病情选择最适合的药物。

抗TREM2抗体的制备及生物学活性的初步研究

目的制备抗髓系细胞触发受体2(TREM2)抗体并初步探究其生物学活性。方法采用杂交瘤筛选的方法,获得稳定分泌抗TREM2抗体的杂交瘤细胞株,并对杂交瘤细胞株分泌的单克隆抗体进行筛选,选择结合效果最佳的单克隆抗体进行人源化改造得到嵌合抗体;对嵌合抗体进行进一步的人源化改造,采用酶联免疫吸附法(ELISA)及流式细胞术(FACS)检测人源化抗体的抗原抗体结合活性;采用生物膜干涉技术(BLI)检测人源化抗体与人TREM2结合动力学;通过基于报告基因的抗体依赖性细胞介导的细胞毒作用(ADCC)生物学活性测定方法,检测人源化抗体的依赖细胞介导的细胞毒效应;使用免疫缺陷小鼠对人源化抗体进行体内药效实验。结果利用杂交瘤技术成功筛选到稳定表达抗TREM2抗体的杂交瘤细胞株,对杂交瘤细胞株进行进一步人源化改造,获得纯度高于95%的人源化抗体h7B6-11,抗体h7B6-11与TREM2-His蛋白具有高度亲和性,且对293T-TREM2细胞的ADCC活性明显强于对照抗体。体内药效实验结果显示,h7B6-11与程序性死亡受体1(PD1)抗体联合用药抑制肿瘤生长效果明显。结论抗体h7B6-11具有高度亲和力和良好的生物学活性,有望开发成为新一代肿瘤免疫治疗的抗体药物。

TCbHP在老年HER-2阳性乳腺癌患者新辅助化疗中的应用效果

目的:研究TCbHP在老年HER-2阳性乳腺癌患者新辅助化疗中的应用效果。方法:选取2020年3月—2022年4月苏州大学附属第三医院收治的100例老年HER-2阳性乳腺癌患者,随机将其分为对照组(n=50)和观察组(n=50)。对照组给予THP新辅助化疗,观察组给予TCbHP新辅助化疗。比较两组临床疗效、治疗前后肿瘤标志物及不良反应。结果:观察组客观缓解率高于对照组,差异有统计学意义(P<0.05)。治疗后,两组癌胚抗原与糖类抗原125水平均低于治疗前,观察组癌胚抗原与糖类抗原125水平均低于对照组,差异有统计学意义(P<0.05)。两组血小板减少、肝损伤、脱发、恶心呕吐、手足综合征发生率比较,差异无统计学意义(P>0.05)。结论:相比THP,TCbHP可进一步抑制老年HER-2阳性乳腺癌患者肿瘤标志物表达,有效控制疾病发展,不会加重化疗引起的不良反应。