人白介素24单克隆抗体的制备与鉴定

目的:制备抗白细胞介素24(Interleukin 24,IL-24)的单克隆抗体并对其生物学特性进行鉴定。方法:应用分子生物学技术,构建含人IL-24编码序列的原核表达载体,表达并纯化了人IL-24融合蛋白。用纯化人IL-24融合蛋白免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用间接ELISA方法筛选杂交瘤细胞,有限稀释法进行亚克隆,采用免疫印迹及免疫组化染色对抗体的特异性进行了鉴定。结果:获得3株能稳定分泌抗IL-24单抗的杂交瘤细胞,分别命名为1G2,3F4,4A6。通过免疫印迹及免疫组化染色检测显示,这3株单抗均能够与IL-24特异性结合。结论:成功制备抗IL-24单克隆抗体,为进一步探索IL-24在抗肿瘤中的作用机制奠定了基础。     

抗p16单克隆抗体的制备及鉴定

目的：制备抗Ｐ１６单克隆工进行鉴定。方法：以Ｐ１６合成肽为抗原免疫纯系小鼠。利用杂交瘤技术制备单克隆抗体,并利用亲和层析等法纯化抗体。通过酶免疫测定及免疫印迹等法对单克隆抗体进行分析及鉴定,并通过免疫组化方法与进我克隆抗体进行比较。结果：获得４株稳定分泌抗Ｐ１６单克隆抗体杂交瘤株,与进口多克隆坑体比较,检测阳性及阴性符合率达１００％,但使用单克隆抗体时,组织切片不需进行抗原修复,染色效果稳定。结论     

肝脏型脂肪酸结合蛋白的重组表达及其单克隆抗体的制备和鉴定

目的： 制备抗肝脏型脂肪酸结合蛋白(LFABP)的单克隆抗体(mAb)并进行亚型和特异性鉴定。方法：以重组的LFABP蛋白为免疫原,经过原核表达和纯化后,免疫BALB/c小鼠,获得分泌鼠抗人LFABP蛋白mAb的杂交瘤细胞株,通过ELISA 和Western blot方法检测其特异性。结果：纯化的LFABP重组蛋白免疫小鼠后经过筛选得到2株稳定分泌抗人LFABP的mAb杂交瘤细胞株,分别命名为3E6和5B7,其亚型分别为IgG1和IgG2a,抗体经纯化后浓度达到2 mg/mL,效价达到1∶10 000以上。Western blot分析结果表明可与细胞中表达的内源LFABP发生特异性免疫反应。结论：成功制备了鼠抗人LFABP的mAb,为进一步研究LFABP的生物学特性,并为相关疾病的治疗奠定了基础。     

抗人微管不稳定蛋白单克隆抗体的制备及鉴定

目的制备人微管不稳定蛋白（Stathmin）的单克隆抗体,检测Stathmin蛋白在真核细胞中的表达,为探讨Stathmin 生物学功能奠定基础。方法利用重组Stathmin蛋白为免疫原,免疫BALB/C小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞Sp2/0进行常规融合,通过间接ELISA的筛选和有限稀释克隆化,获得稳定分泌抗Stathmin单克隆抗体的杂交瘤细胞株,通过ELISA,Western blot和免疫组织化学实验等方法分别对其效价和特异性进行鉴定。结果成功地建立了2株稳定分泌抗Stathmin的单克隆抗体杂交瘤细胞株,分别命名为F001和F002。两株单克隆抗体的免疫球蛋白亚类均为IgG1。两株单克隆抗体通过免疫组织化学实验和Western blot实验都能特异性地结合真核细胞内源性的Stathmin蛋白。结论成功建立了两株效价高、特异性好的抗Stathmin蛋白的单克隆抗体,为进一步研究Stathmin的生物学功能及其与肿瘤的关系创造了条件。     

大肠癌相关新基因ST13单克隆抗体的制备及生物学特性鉴定

目的 研制抗ST13蛋白单克隆抗体,建立检测方法,探讨ST13基因的生物学特性。方法 按淋巴细胞杂交瘤技术制备抗ST13蛋白单克隆抗体,并以亲和层析法纯化单抗,将此单抗作探针用Western-Blot和免疫组化检测标本。结果 获得3个抗ST13蛋白单抗的杂交瘤细胞株,杂交瘤细胞培养上清和腹水中单抗的ELISA效价为10^4-10^5。以ST13单抗作Wester-Blot和免疫组化证实该单抗具有高度特异性,可用于检测组织表达的ST13蛋白。结论 制备了具有高度特异性和高效价的抗ST13蛋白单抗;建立了Western-Blot及免疫组化检测组织表达的ST13蛋白的方法。     

抗上皮细胞黏附分子单克隆抗体的制备及应用

 目的　制备抗人类上皮细胞黏附分子（EpCAM）的单克隆抗体（mAb）并对其进行鉴定及初步功能研究。方法　将原核表达的EpCAM免疫BALB／c小鼠,按常规方法将小鼠脾细胞与小鼠骨髓瘤Sp2／0细胞进行融合,用酶联免疫吸附试验进行阳性克隆的筛选。免疫印迹法对制备的mAb进行鉴定,用免疫组织化学法对临床的3份大肠癌组织样本进行初步检测。结果　酶联免疫吸附试验筛选获得了3株分泌抗EpCAM的mAb杂交瘤细胞株1B10、3G2和4E11。免疫印迹检测结果表明,3株杂交瘤细胞株分泌的抗体均能与EpCAM呈阳性反应,与GST蛋白不发生反应。免疫酶组织化学染色结果显示3株mAb能使3份大肠肿瘤组织样本呈现阳性反应,效果优于对照mAb。结论　成功地制备了3株识别大肠癌细胞表面抗原的mAb,这些mAb在大肠癌的诊断中可能具有一定潜在的应用价值。     

分泌抗人甲胎蛋白单克隆抗体杂交瘤细胞株的建立

应用免疫亲和层析法纯化的正常胎儿组织中的甲胎蛋白(AFP)为抗原免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0采取淋巴细胞杂交瘤技术进行融合,经有限稀释法进行四代克隆化后。获得十株稳定分泌抗人AFP单克隆抗体杂交瘤细胞株。其细胞培养上清中AFP单抗滴度为1:160～640,诱生同系小鼠腹水中单抗滴度为1:4000～100,000。特异性中和抑制试验显示单抗是针对AFP的。放射免疫分析示某些单抗具有较强的亲和力。杂交瘤细胞染色体数目为100～110,其分泌的抗体分别属于IgG_(2a)、IgG_1和IgM型。     

EGFRvⅢ单克隆抗体研制与鉴定

目的:制备针对EGFRvⅢ的单克隆抗体,以期待其对消化道肿瘤的靶向治疗.方法:通过人工合成EGFRvⅢ与KLH连接,免疫Balb/c小鼠,采用杂交瘤技术制备鼠源性单克隆抗体细胞株,经腹水生产抗体,并对抗体进行纯化和相关性质鉴定.结果:合成EGFRvⅢ的基因缺失融合区的相应14肽,短肽与蛋白载体KLH连接作为抗原,免疫Balb/c小鼠.制备针对表达EGFRvⅢ肿瘤而对正常组织无破坏作用的的单克隆抗体.通过常规免疫程序获得高效价(1∶128 000)抗血清的免疫小鼠后,进行细胞融合,制备杂交瘤单克隆抗体细胞.应用ELISA检测的方法筛选鉴定能分泌EGFRvⅢ抗体的杂交瘤细胞株,经亚克隆获得5株鼠源性成系抗体阳性细胞株.阳性细胞株经扩大培养,注射于小鼠腹腔以大量制备抗体.抗体型别鉴定为IgG2a,并对抗体效价及其相对亲合力进行测定.腹水效价达1∶128 000,亲和力最高达9.8×10-9 mol/L.用表达的正常EGFR配体结合区蛋白检测自制抗体的特异性,结果显示抗体不与正常EGFR配体结合区蛋白结合.结论:成功制备了抗鼠EGFRvⅢ单克隆抗体,为后续抗肿瘤的靶向治疗奠定基础.     

肿瘤相关抗原OVA66单克隆抗体的制备和鉴定

目的：制备和鉴定人卵巢癌cDNA文库筛选到的肿瘤相关抗原OVA66的单克隆抗体,为研究其生物学功能提供手段。方法：采用基因重组方法构建pET32bOVA66重组表达质粒,经大肠杆菌诱导表达OVA66融合蛋白,利用NiTED亲和层析技术分离纯化HisOVA66融合蛋白;采用经典的杂交瘤技术制备OVA66单克隆抗体;ELISA和Western blotting鉴定单克隆抗体的生物学和免疫学特性,并对OVA66单克隆抗体在细胞免疫荧光法、流式细胞术和免疫组化等方法中进行了初步应用。结果：成功构建重组表达载体pET32bOVA66,并诱导表达和纯化OVA66融合蛋白。制备获得两株小鼠OVA66单克隆抗体杂交廇细胞株5F4和4G9。两株细胞分泌的抗体亚类均为IgG1,轻链为κ型,亲和力常数Ka分别为2.96×1010和0.4×1010L/mol,初步表位分析结果显示两者针对不同的抗原表位;两株抗体均可以采用细胞免疫荧光和流式细胞术检测OVA66的表达,5F4还可通过免疫组织化学检测肿瘤组织中OVA66的表达。结论：成功制备两株特异性的小鼠OVA66单克隆抗体杂交瘤细胞株,为进一步研究OVA66的生物学特性和临床应用奠定了基础。     

抗CD47单克隆抗体制备及其促巨噬细胞吞噬作用鉴定

目的:制备可识别肿瘤细胞表达CD47蛋白的抗CD47单克隆抗体,并鉴定其抗肿瘤作用。方法:利用BL21(DE3)原核表达系统表达CD47胞外段蛋白,利用Ni-NTA层析纯化表达蛋白。免疫BALB/c小鼠,制备并筛选分泌抗CD47单克隆抗体的杂交瘤细胞株。利用正辛酸-硫酸铵沉淀方法纯化抗CD47单抗。利用流式细胞术检测纯化抗体的活性。利用巨噬细胞与肿瘤细胞共培养试验,检测抗CD47单抗的抗肿瘤作用。结果:表达并纯化了CD47胞外段蛋白,筛选到了分泌抗CD47单克隆抗体的杂交瘤细胞株—7D4,流式细胞术检测结果显示,抗体具有结合肿瘤细胞的活性,但是对不同肿瘤类型细胞的结合效率存在差异。巨噬细胞与肿瘤细胞共培养试验发现,利用抗体封闭肿瘤细胞CD47信号后,可以增强巨噬细胞对肿瘤细胞的吞噬作用。结论:抗CD47单抗—7D4可以促进巨噬细胞的吞噬作用,具有潜在应用价值。     

抗p53融合蛋白单克隆抗体的性质鉴定

目的 :研制出能特异与p5 3结合的单克隆抗体 ,使对 p5 3的检测得到更为广泛的应用。 方法 :以原核表达的 p5 3-GST融合蛋白为免疫原 ,常规方法作细胞融合 ,间接酶联免疫吸附试验 (ELISA)双筛和免疫组织化学(IHC)筛选 ,将能稳定分泌抗人p5 3单抗的杂交瘤细胞株的细胞上清 (命名为M12 6 )与常用p5 3进口单抗PAB180 1(ZYMED公司 )作对比实验。结果 :此单抗适用于ELISA、IHC、免疫印迹及免疫沉淀等方面的研究 ,并且在一定程度上优于PAB180 1。结论 :新制备的单抗M12 6可考虑取代进口单抗PAB180 1而用于p5 3的表达及突变研究。     

Preparation and preliminary characterization of rabbit monoclonal antibodies against human midkine

We prepared rabbit monoclonal antibodies that target human midkine (MK). The MK gene was amplified by PCR from the plasmid pEGFP-MK and subcloned into the prokaryotic expression vector pGEX-1λT to generate an N-terminally glutathione S-transferase (GST)-tagged fusion protein construct. Expression of the GST-MK fusion protein was achieved by IPTG induction in Escherichia coli cells. The expressed protein was purified using the GST system. After verifying purification, the fusion protein was used to immunize rabbits to prepare monoclonal antibodies against human MK by the rabbit hybridoma technique. The hybridomas generated were screened by an enzyme-link immunoassay (ELISA) for specificity, which was further characterized by Western blotting and ELISA. SDS-PAGE analysis showed that the purified protein corresponds to the calculated molecular weight. The GST-MK fusion protein was prepared. At least one hybridoma cell line secreting anti-MK MAb was obtained. Western blotting analysis confirmed the identity of the MAb. The titer of the MAbs measured by an indirect ELISA was 1:64,000. The affinity constant, which was measured by a non-competitive ELISA, was found to be 3.0?×?10(9) M(-1). Western blotting and immunohistochemistry analysis showed that the produced MAbs bind to the MK protein in cancerous tissues. The GST-MK fusion protein was successfully expressed and purified. The MAbs against MK were subsequently prepared, which should further aid research and the application of MK MAbs in clinical settings.     

Monoclonal antibodies against human tumor metastasis suppressor gene-1 (TMSG-1): preparation, characterization, and application

To better understand the molecular mechanism of metastasis, the monoclonal antibody against tumor metastasis suppressor gene-1 (TMSG-1, one novel gene) was prepared, characterized, and applied in estimating the metastatic potential of human tumors. A dominant epitope, TMSG-1(15)--derived from TMSG-1--was automatically synthesized based on the Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen solution was used to immunize BALB/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and their characterization was performed by Western blotting and immunohistochemical staining. The hybridoma cell line secreting anti-TMSG-1 antibody, designated C8, was established after primary ELISA screening and consequent rapid limited dilution. C8 was IgM in isotyping. The competitive inhibition assay showed that the antibody was TMSG-1 specific. Furthermore, in Western blotting, a protein band of about 45 kD was detected with nonmetastatic variant PC-3M-2B4 and PG-LH7 cells, but not with the isogenetic metastatic variant PC-3M-1E8 and PG-BE1 cells. Immunohistochemistry method showed that positive staining presented in the cytomembrane and cytoplasm of 2B4 and LH7 cells, while 1E8 and BE1 cells were non-reactive. The sections from paraffin-embedded blocks of human cancer (52 cases of breast carcinoma and 41 cases of colon cancer) were stained, showing strongly positive in non-metastatic tumor and weakly positive or negative in metastatic tumor; the strongly positive rate of TMSG-1 expression in human cancers were, respectively, 36%, 7.4%, 52.4%, and 35% in non-metastatic and metastatic breast cancer and non-metastatic and metastatic colon cancer (p = 0.02). The monoclonal antibody developed against synthetic peptide was TMSG-1 specific, and it has promise in Western blot and immunohistochemistry for detecting the TMSG-1 expression of cancer cells and tissues. It may provide an important tool in the study of TMSG-1 expression and function in both experimental and clinical studies. Furthermore, our tests confirmed that TMSG-1 protein has excellent inverse correlation to tumor metastasis potential, with the molecular weight of 45 kD (supporting the encoded protein containing 380 amino acids) and localization in cytomembrane and cytoplasm of tumor cell.     

抗人双特异性蛋白磷酸化酶18单克隆抗体的制备及鉴定

目的制备高亲和力、高特异性的抗人双特异性磷酸化酶18（Dusp18）单克隆抗体,并对其生物学特性进行鉴定,为Dusp18功能的研究奠定基础。方法重组Dusp18免疫BALB／c小鼠,取其脾细胞与SP2／0细胞融合,经筛选建立可稳定分泌抗Dusp18的单抗细胞株,制备腹腔积液,ProteinG纯化所得腹腔积液,ELISA及Westernblotting检测抗体的效价和亲和力,用亚型鉴定试纸条鉴定单克隆抗体的亚型。应用免疫组织化学检测Dusp18在肝癌组织中的表达情况。结果获得两株（F003和F004）单抗,上清效价分别为1：5120和1：10240,腹腔积液效价分别为1：25600和1：51200,Westernblotting结果均在预期位置出现阳性条带。利用两株抗体均可在肝癌组织中检测到Dusp18的表达。两株抗体亚型均为IgG1型,轻链均为k型。结论成功制备能特异性识别Dusp18的单克隆抗体,为进一步研究Dusp1的生物学功能,揭示其与肿瘤发生、发展之间的关系奠定了基础。     

抗人骨唾液蛋白单克隆抗体的制备及鉴定

目的制备高亲和力、高特异性的抗人骨唾液蛋白（BSP）单克隆抗体（mAb）,并对其生物学特性进行鉴定,为BSP作为乳腺癌骨转移临床诊断靶点的研究奠定基础。方法重组BSP蛋白免疫BALB／c小鼠,取其脾细胞与Sp2／0细胞融合,经筛选建立可稳定分泌抗BSPmAb细胞株,制备腹腔积液,ProteinG纯化。ELISA检测抗体的效价和亲和力,并用亚型鉴定试纸条鉴定mAb的亚型。应用Western blotting检测mAb的特异性,进一步利用纯化的mAb经免疫组织化学法检测乳腺癌细胞MDA-MB-231中BSP的表达情况。结果获得9株抗BSPmAb细胞株,选择其中2株（D001和D002）进一步鉴定,其上清效价分别为1：5120和1：10240,腹腔积液效价分别为1：25600和1：51200。2株抗体亚型均为IgG1型,轻链均为K型。Western blotting结果均在预期位置出现阳性条带。利用此2株细胞株所得抗体均可在乳腺癌细胞MDA-MB-231中检测到BSP的表达。结论成功制备能特异性识别BSP的mAb,为进一步研究BSP的生物学功能以及对BSP作为乳腺癌骨转移的标志物进行临床评价奠定基础。