马铃薯卷叶病毒单克隆抗体的制备

采用杂交瘤技术,以马铃薯卷叶病毒(Potato Leafroll Virns,PLRV)为抗原,用直接将病毒注入脾脏和随后尾静脉注射的方法,免疫BALB/C小鼠。将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0融合。用Dot-ELISA和间接血凝试验筛选分泌抗马铃薯卷叶病毒抗体的阳性克隆,建立了分泌抗PLRV单克隆抗体的杂交瘤细胞株。用微量玻片双扩散法测定单克隆抗体亚类为IgG_1和IgG2a,轻链为λ。注射杂交瘤细胞株A_1、A_3、C_3和D_3于小鼠腹腔,制备出含高效价单克隆抗体的腹水。用获得的四种单克隆抗体对马铃薯卷叶病毒15个分离物进行了鉴定。     

蛋白质芯片技术应用于高通量单克隆抗体制备研究

针对在传统的单克隆抗体制备过程中进行特异性筛选时大量的人力消耗,建立了一种联合应用蛋白质芯片进行单克隆抗体制备的方法。用8种重组蛋白分别免疫BALB/c小鼠,在传统的细胞融合的基础上,将8种抗原免疫的杂交瘤阳性细胞混合后进行克隆化、蛋白质芯片筛选,阳性细胞有限稀释克隆化制备相关抗体。实验结果：混合克隆化共得到单克隆细胞175孔,经蛋白质芯片筛选出阳性孔119孔,选择针对单一抗原阳性的细胞连续2轮克隆化,8种重组蛋白各获得单克隆抗体细胞株1株。与经典的单克隆抗体制备相比,蛋白质芯片筛选与混合克隆化技术联合应用于单克隆抗体制备,1个筛选周期获得了8种重组蛋白的单克隆抗体细胞株,提高了单克隆抗体的制备效率,节省了在筛选中的抗原用量,提供了一种经济、快速、简便的方法。     

蛋白质芯片技术应用于高通量单克隆抗体制备研究

针对在传统的单克隆抗体制备过程中进行特异性筛选时大量的人力消耗,建立了一种联合应用蛋白质芯片进行单克隆抗体制备的方法。用8种重组蛋白分别免疫BALB/c小鼠,在传统的细胞融合的基础上,将8种抗原免疫的杂交瘤阳性细胞混合后进行克隆化、蛋白质芯片筛选,阳性细胞有限稀释克隆化制备相关抗体。实验结果:混合克隆化共得到单克隆细胞175孔,经蛋白质芯片筛选出阳性孔119孔,选择针对单一抗原阳性的细胞连续2轮克隆化,8种重组蛋白各获得单克隆抗体细胞株1株。与经典的单克隆抗体制备相比,蛋白质芯片筛选与混合克隆化技术联合应用于单克隆抗体制备,1个筛选周期获得了8种重组蛋白的单克隆抗体细胞株,提高了单克隆抗体的制备效率,节省了在筛选中的抗原用量,提供了一种经济、快速、简便的方法。     

抗人B7-H1单克隆抗体的制备和鉴定

目的:采用杂交瘤技术制备抗人B7-H1单克隆抗体,并对其进行鉴定。方法:经抗原免疫的小鼠脾细胞与小鼠骨髓瘤细胞以常规方法融合;用间接ELISA法筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法获得稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注射进小鼠腹腔后制备腹水;纯化腹水中的单克隆抗体并对其亚型进行鉴定;用间接ELISA法测抗体效价;将肺癌组织制成石蜡切片,用抗人B7-H1抗体进行免疫组化染色。结果:获得1株稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株,所分泌的单抗类型为IgG1;抗体效价为1×108,纯化后的抗体含量为6.76g/L;免疫组化实验中,单抗可与肺癌组织表面的B7-H1蛋白特异地结合。结论:制备了人B7-H1单克隆抗体,为B7-H1检测试剂盒的研制奠定了基础。     

探究杂交瘤技术制备单克隆抗体时与骨髓瘤细胞有效融合的细胞类型

杂交瘤技术制备单克隆抗体,一般取免疫小鼠的脾细胞与骨髓瘤细胞融合,并通过筛选获得杂交瘤细胞后再生产。脾脏内含有B1细胞、Mz-B细胞、T1 B细胞、T2 B细胞、初始B细胞、致敏B细胞、短寿命浆细胞、中心母细胞、中心细胞、抗体分泌细胞等不同类型的细胞。制备单克隆抗体使用的抗原多为蛋白质,经典的免疫策略要用抗原反复刺激免疫动物,所获单克隆抗体类型多为高亲和力的免疫球蛋白G(IgG),结合近期发明的一些新技术等,可认为与骨髓瘤细胞有效融合的主要是由记忆B细胞增殖分化而来的抗体分泌细胞。     

金黄色葡萄球菌肠毒素C3单克隆抗体的制备与鉴定

目的制备稳定分泌抗金黄色葡萄球菌肠毒素C3（SEC3）单克隆抗体的杂交瘤细胞株,并对单克隆抗体的性质进行鉴定。方法以SEC3重组蛋白免疫Balb／c小鼠,应用细胞融合技术将小鼠的脾细胞与sR／0骨髓瘤细胞进行融合,经间接ELISA法检测筛选及2次有限稀释法克隆化培养,获得目的杂交瘤细胞株,并对其所产生的单克隆抗体进行效价、亲和常数及抗原识别表位等相关性质的鉴定。结果最终获得了两株能分泌单克隆抗体的杂交瘤细胞1C12和2A2,两者细胞培养上清的效价分别为1：3200和1：1600。经分析可知1C12细胞株的亲和力高于2A2细胞株,同时相加实验表明两个单克隆抗体识别抗原表位相同。结论单克隆抗体制备成功,为进一步完善肠毒素SEC3的临床检测奠定了基础。     

杂交瘤细胞体外大规模培养研究进展

单克隆抗体在生物学和医学研究领域中显示了极大的应用价值,是免疫检验中的新型试剂,是生物治疗的导向武器。作为医学检验试剂,单克隆抗体可以充分发挥其优势,如特异性好,灵敏度高,更便于质量控制,利于标准化和规范化。传统的方法是利用小鼠腹水制备单克隆抗体,但是近几十年杂交瘤细胞体外大规模培养制备单克隆抗体技术也在不断发展。特别是单克隆抗体在疾病诊断和治疗方面的需求,更进一步促进了杂交瘤细胞体外培养生产技术的发展,体外培养杂交瘤细胞生产的单克隆抗体已应用到许多方面。由于杂交瘤细胞的半贴壁性质,无论是悬浮培养还是贴壁培养,均可进行杂交瘤细胞的体外大规模培养。针对应用于体外诊断试剂的杂交瘤细胞体外培养制备单克隆抗体进行综述,主要包括中空纤维细胞培养和生物反应器细胞培养方法,以及不同培养方法优化的进展。     

人凝血因子Ⅶ单克隆抗体的制备与鉴定

目的：制备抗人凝血因子Ⅶ单克隆抗体并鉴定其特性。方法：应用杂交瘤融合技术,以重组人凝血因子Ⅶ为抗原免疫BALB／c小鼠;取免疫小鼠的脾细胞与Sp2／0骨髓瘤细胞融合,经间接ELISA法筛选、融合细胞有限稀释法克隆、克隆化杂交瘤细胞株的亚类鉴定等方法筛选出单克隆抗体杂交瘤细胞株,并对单克隆抗体的特异性进行鉴定;用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行单抗的纯化。结果：获得了3株可稳定分泌单克隆抗体的杂交瘤细胞3E8、3D2和1C5,诱生的腹水效价分别为1：1×10^7、1：1×10^6和1：1×10^6;亚类鉴定表明388为IgG2a,其余2株均为IgGl;特异性鉴定显示它们与多种血浆蛋白均无交叉反应,表明单抗是特异的;经过亲和层析,获得了纯化的单抗。结论：获得了特异性的人凝血因子Ⅶ单克隆抗体,为建立人凝血因子Ⅶ检测及纯化方法奠定了基础。     

抗重组人表皮生长因子单克隆抗体的制备及生物学特性鉴定

建立能稳定分泌特异性抗重组人表皮生长因子(rhEGF)单克隆抗体的杂交瘤细胞株 ,制备出高效价的 ,具有生物学活性的抗重组人表皮生长因子单克隆抗体 ,为EGF及其单克隆抗体在肿瘤生长调节中的作用研究 ,为肿瘤的诊断与治疗提供具有实用价值的研究工具。此外 ,为EGF的亲和层析纯化提供材料。以BALB/c小鼠脾细胞为饲养细胞 ,运用细胞杂交瘤技术制备 ,间接ELISA法筛选出能稳定分泌针对人表皮生长因子的单克隆抗体细胞株 ;对杂交瘤细胞进行染色体核型分析并以体内诱生法产生腹水以获得大量单克隆抗体 ,并采用HiTrapProteinAHP柱对其纯化…     

番茄环斑病毒单克隆抗体的制备

以纯化的番茄环斑病毒(Tomato ringspot virus,ToRSV)为抗原,注射免疫BALB/c小鼠,将免疫小鼠脾细胞与小鼠骨髓瘤细胞Sp2/0进行融合,经多次细胞筛选及克隆化,获得3株(A8、B7和G9)可分泌抗ToRSV单克隆抗体的杂交瘤细胞株,并以之分别制备小鼠腹水单克隆抗体。经酶联免疫吸附试验检测表明,该3株杂交瘤细胞腹水抗体效价在10-5～10-6之间,且均具有与ToRSV反应的特异性。     

Preparation of human salivary alpha-amylase specific monoclonal antibody

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.     

Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection

Antibodies are very important materials for diagnostics. A rapid and simple hybridoma screening method will help in delivering specific monoclonal antibodies. In this study, we systematically developed the first antibody array to screen for bacteria-specific monoclonal antibodies using Listeria monocytogenes as a bacteria model. The antibody array was developed to expedite the hybridoma screening process by printing hybridoma supernatants on a glass slide coated with an antigen of interest. This screening method is based on the binding ability of supernatants to the coated antigen. The bound supernatants were detected by a fluorescently labeled anti-mouse immunoglobulin. Conditions (slide types, coating, spotting, and blocking buffers) for antibody array construction were optimized. To demonstrate its usefulness, antibody array was used to screen a sample set of 96 hybridoma supernatants in comparison to ELISA. Most of the positive results identified by ELISA and antibody array methods were in agreement except for those with low signals that were undetectable by antibody array. Hybridoma supernatants were further characterized with surface plasmon resonance to obtain additional data on the characteristics of each selected clone. While the antibody array was slightly less sensitive than ELISA, a much faster and lower cost procedure to screen clones against multiple antigens has been demonstrated.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

庆大霉素单克隆抗体的制备及试剂盒的配制

目的建立庆大霉素直接竞争酶联免疫吸附分析方法。方法应用戊二醛法制备庆大霉素完全抗原,通过杂交瘤技术筛选分泌特异性庆大霉素抗体的杂交瘤细胞株,并建立庆大霉素竞争酶联免疫吸附分析检测方法。结果获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,建立了庆大霉素竞争酶联免疫吸附分析检测方法,该方法操作简单具有良好的线性、特异性和精密度;庆大霉素质量浓度在1.5625～50.0000 ng/mL范围内,呈现良好的线性,r2=0.9913,50%抑制浓度为(IC50)为7.37 ng/mL,检测限(LOD)为1.54 ng/mL,该试剂盒与链霉素等8种药物无交叉反应。结论获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,研制的庆大霉素竞争ELISA检测试剂盒具有良好的线性、特异性和精密度。     

A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas

B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.     

Large-scale production of monoclonal antibodies in defined serum-free media in airlift bioreactors

Forty- and ninety-liter airlift bioreactors have been used successfully to grow hybridoma cell lines in chemically defined serum-free media. In the airlift bioreactor, hybridoma cell growth and monoclonal antibody productivity are comparable to that obtained by conventional cell culture. At sparging rates of 0.60-1.20 vvh (volume of sparged gas per bioreactor volume per hour), the airlift bioreactor achieves rapid mixing and adequate oxygen mass transfer. Foaming is minimal and inconsequential for serum-free media and media supplemented with 5%-10% fetal bovine serum. The use of serum-free medium facilitates monoclonal antibody purification and enhances the purity of the final MAb product.     

A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay

In hybridoma screening, quantitative kinetic evaluation is difficult since the concentration of each antibody in the hybridoma supernatant is unknown. From modeling calculations, we hypothesized that the ratio of two different antigen-antibody concentrations might allow discrimination of high-affinity monoclonal antibodies irrespective of the antibody concentration. Using anti-alpha-fetoprotein monoclonal antibodies of known affinity, we set the signal ratio of a time-resolved assay at >0.1, in which the antigen concentrations were 10 and 100 ng/mL. From anti-alpha-fetoprotein hybridoma screening with this assay, it was possible to effectively select high-affinity monoclonal antibodies with KD values below 1x10(-8) M. High-sensitivity sandwich enzyme-linked immunosorbent assay which detects domain III of alpha-fetoprotein has been established using selected high-affinity monoclonal antibodies. This screening method is useful for selection of high-affinity monoclonal antibodies of potential diagnostic value.     

筛选抗甲3型（H3N2）流感病毒单克隆抗体ELISA间接方法的建立

用血凝抑制实验方法,虽可直接筛选到抗甲_3型流感病毒血凝素(H)单克隆抗体,但检测出的杂交瘤培养物上清中有小牛血清,做血凝抑制时还需用受体破坏酶处理去除非特异性抑制物。为减少麻烦我们建立了便于大批检测和筛选抗甲_3型(H_3N_2)流感病毒单克隆抗体的ELISA间接方法。