两株抗肝癌人-鼠杂交瘤细胞系的建立及初步鉴定

目的建立两株产生人源性抗肝癌单克隆抗体的人-鼠杂交瘤.方法用IL-2和美洲商陆(PWM)体外刺激肝癌病人外周血淋巴细胞再与小鼠骨髓NS-1融合,待杂交瘤生长进行筛选、克隆化及特性分析.结果建立了两株产生人源性抗肝癌单克隆抗体的人-鼠杂交瘤AF3、CB7.二种单抗均识别肝癌细胞表面抗原,并具有显著的补体依赖的细胞毒活性.其中AF3的效价最高.结论通过人一鼠杂交瘤技术获得了产生人源性抗肝癌单克隆抗体的杂交瘤细胞系.抗体效价高,是一种较理想的单克隆抗体.     

HER2抗独特型单克隆抗体的制备和初步研究

目的:研制HER2抗独特型单克隆抗体,为进一步深入研究乳腺癌抗独特型抗体疫苗奠定基础。方法:用人HER2蛋白免疫家兔,获得特异性兔抗HER2抗体。再用兔抗HER2抗体免疫Balb/c小鼠,采用杂交瘤技术制备HER2抗独特型单克隆抗体。并筛选出β型HER2抗独特型单克隆抗体。结果:ELISA检测结果表明,获得的兔抗HER2抗体能特异性地与HER2蛋白结合,其OD值随HER2的浓度呈正线性关系。用兔抗HER2抗体免疫小鼠获得一株稳定分泌HER2抗独特型单克隆抗体的杂交瘤细胞1F5,其分泌的单克隆抗体能特异性的和兔抗HER2多克隆抗体结合,并与HER2产生竞争抑制作用。用1F5抗独特型单克隆抗体免疫家兔得到的抗血清能和HER2特异性的结合。该抗体亚型为IgG3,未浓缩腹水的效价为1∶1.02×104。结论:抗独特型单克隆抗体为β型HER2抗独特型抗体,有可能成为乳腺癌抗独特型单克隆抗体疫苗。     

人源性抗肝癌单克隆抗体的制备及初步鉴定

目的建立产生人源性抗肝癌单克隆抗体的人-鼠杂交瘤.方法用IL-2和美洲商陆(PWM)体外刺激肝癌病人外周血淋巴细胞,再与小鼠骨髓瘤细胞NS-1融合,待杂交瘤生长进行筛选、克隆化及特性分析.结果建立了3株产生人源性抗肝癌单克隆抗体的人-鼠杂交瘤-AF3,CB7、CH1.三种单抗均识别肝癌细胞表面抗原,并具有显著的补体依赖的细胞毒活性.其中AF3的效价最高.结论通过人-鼠杂交瘤技术获得了产生人源性抗肝癌单克隆抗体的杂交瘤细胞系.抗体效价高,是一种较理想的单克隆抗体.     

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     

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。     

大肠癌患者血清中巨噬细胞抑制因子-1升高的临床价值研究

目的 探讨巨噬细胞抑制因子1(MIC-1)在大肠癌诊断和早期诊断、治疗监测及复发预警中的临床价值.方法 应用自主研制的MIC-1检测试剂盒检测239例不同临床分期的大肠癌患者、16例肠道良性疾病患者及200例健康人血清样本中MIC-1水平,并对部分肿瘤患者进行连续监测;应用罗氏Cobas 601电化学发光免疫分析仪检测...     

胃癌患者血清中巨噬细胞抑制因子-1的临床价值研究

目的 探讨巨噬细胞抑制因子1(MIC-1)在胃癌诊断、治疗监测及复发预警中的临床价值.方法 应用自主研制的MIC-1检测试剂盒检测179例不同临床分期的胃癌患者、9例胃部良性疾病患者及200例健康人群血清样本中MIC-1水平,并对部分肿瘤患者进行动态监测;同时应用罗氏Cobas 601电化学发光免疫分析仪检测上述样品的...     

抗甲状腺球蛋白单克隆抗体的制备及应用

目的:制备分泌特异性抗人甲状腺球蛋白(Tg)单克隆抗体的杂交瘤,建立检测Tg的ELISA方法。方法:以人甲状腺球蛋白为抗原免疫BALB/c小鼠,通过细胞融合制备分泌抗人TgmAb,并对其进行特异性鉴定,建立双抗体ELISA夹心法检测Tg。结果:共获得7株可稳定分泌抗人TgmAb的杂交瘤细胞株FMU-TG1～FMU-TG7,用单抗FMU-TG和Tg多克隆抗体建立了检测人Tg夹心ELISA试剂盒,敏感性可达30ng/ml,结论:成功制备了抗人TgmAb并建立了检测人Tg夹心ELISA试剂盒,对Tg的基础研究及临床应用具有重要意义。     

胰腺癌患者血清中巨噬细胞抑制因子-1的大样本临床研究

目的 探讨巨噬细胞抑制因子1(MIC-1)在胰腺癌诊断、早期诊断及治疗监测中的临床价值.方法 应用自主研制的MIC-1检测试剂盒检测552例不同临床分期的胰腺癌患者、115例胰腺良性肿瘤患者、21例慢性胰腺炎及200例健康人血清样本中MIC-1水平,并对部分早期肿瘤患者的病程进行随访监测;应用罗氏Cobas 601电化...     

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。     

抗腺病毒载体单克隆抗体的制备及其初步鉴定

Objective:To prepare and identify monoclonal antibodies(McAbs)against the capsid proteins of adenovirus vector.Methods:BALB/c mice were immunized with a mixture of the purified adenovirus vector(Adv)and Al(OH)3.McAbs were produced using cell fusion technique in a conventional way.The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay(ELISA),immunocytochemical staining and Western blotting. Results:Six strains of hybridoma cells(A4H11,A8C7,F1H5,G1D2,G4E3 and H2G8)that can stably secrete the IgG1 McAb against Adv were obtained.After 3 months subculture and low concentration of serum adapting culture,six strains retained their stability to secrete McAb.The ascites titers were between 1:106 and 1:108.Western blot analysis demonstrated that all the McAbs reacted with one protein(about 114 kDa)which is present in wild type 3 adenovirus(wtAd3),wild type 5 adenovirus (wtAd5),wild type 7 adenovirus(wtAd7)and adenovirus vector.Conclusion:Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector,and provided the substantial foundation of preclinical research of adenovirus vectors.     

Production of monoclonal and polyclonal antibodies against prostate-specific antigen, a prostate cancer serum marker

The aim of this study was to produce monoclonal and polyclonal antibodies against prostate-specific antigen (PSA), a prostate cancer serum marker. Hyperimmune ICR mice produced polyclonal antibodies (PoAbs) after injection with 0.5 mL of pristane, and were injected with NS-1 myeloma cells 2 weeks later. Hyperimmune Balb/c mice were used for the production of monoclonal antibodies (MAbs). Mice were immunized four times and given a final boost, and their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500. The fused cells were then selected in the HAT-RPMIX medium. Anti-PSA antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS) HT-RPMIX medium. Twelve murine hybridoma producing anti-PSA MAbs were obtained and designated C3m1G11, B3m1E5, C3m1E8, C3m1C5, C3m2F4, C3m1F8, C3m2B3, C3m2E6, B3m2B11, B3m2F2, C3m2C7, and C3m2D9. Isotypes of these MAbs were identified as IgG2a heavy chain and kappa light chain. Hitrap Protein A column was used for the purification of polyclonal and monoclonal antibodies. The purity analysis of MAb was performed by capillary electrophoresis.     

Production and purification of monoclonal and polyclonal antibodies against cholera toxin

The aim of this study was to produce monoclonal and polyclonal antibodies against cholera toxin (CT). Hyperimmune ICR mice produced polyclonal antibodies (PAbs) after injection with 0.5 mL of pristane and were injected with NS-1 myeloma cells 2 weeks later. Hyperimmune Balb/c mice were used for the production of monoclonal antibodies (MAbs). After these mice were immunized four times and given a final boost, their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500. The fused cells were then selected in the hypoxanthine, aminopterin, and thymidine (HAT)-RPMIX medium. Anti-CT antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS) HT-RPMIX medium. Eleven murine hybridoma producing anti-CT MAbs were obtained and designated CT-A2, CT-B4, CT-B11, CT-C7, CT-D7, CT-E8, CT-F4, CT-F2, CT-F8, CT-E3, CT-E6. Isotypes of MAbs were identified as IgM heavy chain and all were lambda light chain. Hitrap rProtein A and Hitrap IgM purification columns were used for the purification of PAbs and MAbs, respectively.     

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。     

Generation of anti-idiotypic antibodies that mimic HBsAg and vaccination against hepatitis B virus

The advent of hybridoma technology has opened up a new avenue in vaccine development, and antigen-mimicking properties of anti-idiotypic antibodies have provided promising alternatives in the generation of experimental anti-idiotypic vaccines. In the present study, mice were immunized with anti-hepatitis B virus (HBV) mouse monoclonal and anti-HBV goat polyclonal antibody to produce anti-idiotypic antibodies. Two mouse monoclonal antibodies (6C9, 6H9) were obtained from the fusions, and the immunogenic properties and specificity of antibodies were analyzed. BALB/c mice were immunized with varying concentrations of anti-idiotypic antibodies (25, 50, 75, and 100 micrograms of anti-Id), and it was shown that anti-idiotypic antibodies generated hepatitis B surface antigen (HBsAg), as well as a BSA-specific antibody response. A simple method for the purification of monoclonal antibodies by dialyzing antibody against water has also been reported.     

Preparation and identification of monoclonal antibodies against daintain

Daintain is a 17-kDa polypeptide originally purified from porcine intestine. This polypeptide is associated with insulin secretion and inflammatory responses. Daintain is highly similar in amino acid sequence to allograft inflammatory factor-1 (AIF-1). Here we report the preparation and identification of monoclonal antibodies (MAbs) against daintain. To enhance its immunogenicity, daintain was coupled to carrier protein bovine serum albumin (BSA) by a two-step glutaraldehyde method. Using conventional procedures, we obtained four stable hybridoma cell lines that can produce and secret anti-daintain MAbs. We further analyzed their isotypes, titer, and affinity, and found that those MAbs belong to the G1 subclass with kappa light chains. The MAbs were capable of recognizing daintain as determined by Western blotting. The produced MAbs will be a useful tool for further investigation of daintain functions in organisms.     

Study of anticancer human monoclonal antibody--establishment of human monoclonal antibody to gastric cancer by human-mouse hybridoma

A human immunoglobulin M (IgM) antibody secreting hybridoma, HMG1, has been established and studied for its reactivity against human gastric cancer cells. Lymphocytes isolated from a regional lymph node of patient with gastric adenocarcinoma were fused with mouse myeloma cells NS-1. Supernatants from the generated human-mouse hybrids were first screened for immunoglobulin production by ELISA. The identified human IgM-secreting hybridomas were expanded and subcloned for further analysis or cryopreservation. The screening for binding of antibodies to a panel of human cancer cell lines and normal fibroblast was carried out with PAP or indirect immunofluorescence stain. The selected hybridoma, HMG1 after being cloned three times, was stable in secreting IgM (about 4 micrograms/1 x 10(6) cells) for more than 9 months. Large amount of ascites was obtained by injecting this hybrid to BALB/C nude mice pretreated with anti-lymphocyte serum and pristane. The ascitic fluid contained 5-19 mg human Ig/ml. Subsequently this IgM was extracted from ascitic fluid by saturated ammonium sulphate solution. This crude extract was further purified with immuno-affinity chromatography. Both this purified ascite-IgM as well as IgM from HMG1 supernatant would react with gastric cancer cell line BGC-823 but not with human normal fibroblasts 350Q by PAP or immunofluorescence analysis. The human HMG1-IgM reacted with gastric cancer cells on paraffin embedded tissue section but did not react with normal gastric mucosa cells. HMG1-IgM had some complement dependent cytotoxicity against BGC-823. These results suggest that the establishment of anticancer human monoclonal antibodies with human-mouse hybridoma technique be feasible. There is a possibility for clinical applications of this human monoclonal antibody in the future.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human monoclonal antibodies directed against melanoma tumor-associated antigens

To analyze the humoral immune response to melanoma, human-mouse hybridomas were generated by the fusions of regional lymph node lymphocytes of patients with the mouse myeloma cell line M5. Six stable hybridomas were cloned from six separate lymphocyte parents obtained from three patients. Ascites were obtained from nude mice after i.p. injection with cultured hybridoma cells. The monoclonal antibodies, four immunoglobulin Gs and two pentameric immunoglobulin Ms, were partially purified to remove mouse immunoglobulin and then conjugated to biotin for immunocytochemical and immunohistochemical studies. With the avidin:biotin:peroxidase complex method to detect and amplify binding by the biotin-conjugated human monoclonal antibodies, we found the six antibodies to be reactive against cytoplasmic determinants in five short-term melanoma cultures and formalin-fixed paraffin-embedded melanoma tumors from four patients. The antigenic target of the antibodies identified was not carcinoembryonic antigen. Two antibodies, 2-139-1 and 6-26-3, were studied in more detail. Each stained 25 of 25 specimens of melanomas. Little or no reactivity was detected against fixed sections of normal skin, which included tissues such as epidermis, dermis, monocytes, lymphocytes, and vascular endothelium. More striking was the absence of binding to melanocytes in the basal layer of the skin or to pigmented nevus cells. Both antibodies showed cross-reactivity against other tumors, in particular colonic and prostatic carcinomas. In the normal colon, reactivity was restricted to the surface of the columnar epithelium; no reactivity was detected against normal prostatic epithelium. Reactivity was also not observed against liver and lung. However, the epithelia of the renal tubules, pancreatic ducts, and salivary ducts were all reactive. These human monoclonal antibodies identify cytoplasmic melanoma-associated tumor antigens that appear different from the membrane antigens defined by serological approaches and by most mouse monoclonal antibodies.     

Development of a monoclonal antibody against glucosyltransferase D of Streptococcus mutans GS 5

Glucosyltransferases GtfB, GtfC, and GtfD of Streptococcus mutans are virulent factors involved in dental caries. Consequently, they are considered to be target molecules in the development of vaccines against dental caries. Among them, GtfD plays a significant role in the sucrose-dependent cellular adhesion of S. mutans, and a number of studies have suggested that the N-terminus of GtfD is an important part of its role in enzymatic activity. In this study, we generated monoclonal antibodies against the N-terminus of GtfD (anti-GtfDN antibody) in an initial attempt to investigate its preventive efficacy against dental caries. To obtain anti-GtfDN monoclonal antibodies, the gene for the N-terminus of gtfD (2?kb) was cloned into an Escherichia coli expression vector, pQE30; then the expressed protein (about 75?kDa) was purified. The purified GtfDN protein was injected into BALB/c mice, and hybridoma clones were established. We obtained three hybridoma clones (HDN9, HDN11, and HDN28) capable of producing anti-GtfDN antibodies. Their binding specificity was characterized by ELISA, dot blot, and Western blot analysis after purification using affinity column chromatography. The isotype of the monoclonal antibodies was confirmed to be IgG2a.