纯化和标记抗人轻链β2m单克隆抗体的方法

背景：通过杂交瘤细胞株接种小鼠腹腔可获得含大量抗体的腹水,但以往纯化腹水中单克隆抗的方法较复杂,不易操作。目的：制备、纯化和标记抗人类白细胞抗原Ⅰ类分子轻链的单克隆抗体,以检测肿瘤细胞表面的人类白细胞抗原Ⅰ类分子的表达。方法：将杂交瘤细胞接种至小鼠腹腔,获得含抗人轻链β2m抗体的腹水,用改良的辛酸-硫酸铵方法纯化腹水,将纯化后的单克隆抗体标记异硫氰酸荧光素（FITC）,标记后的抗体用于检测外周血单个核细胞、表达空载人类白细胞抗原A2分子的T2细胞和白血病K562细胞表面的人类白细胞抗原Ⅰ类分子,并用流式细胞仪和荧光显微镜观察人类白细胞抗原Ⅰ类分子的表达。结果与结论：纯化后的抗人轻链β2m-FITC单克隆抗体纯度为96%。流式细胞检测结果表明,人类白细胞抗原Ⅰ类分子在外周血单个核细胞表面高表达,在表达空载人类白细胞抗原A2分子的T2细胞表面低表达,而在白血病K562细胞表面不表达。结果证实,用改良的辛酸-硫酸铵方法纯化腹水以此制备的抗人轻链β2m-FITC能有效区别不同细胞表达人类白细胞抗原Ⅰ类分子的强弱,其纯化方法简便易行。     

抗重组人白细胞介素1β单克隆抗体识别相应抗原决定簇的研究

本文对３个抗重组人白细胞介素１β（ｒＨＩＬ－１β）单克隆抗体识别相应抗原决定族的特性进行了研究。Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ结果证明：Ｂ３，Ａ７，Ｅ５３个单克隆抗体识别分子量为１７．５ｋＤ的ＩＬ－１β。竞争性ＥＬＩＳＡ证实，３个抗体识别ＩＬ－β分子上两个抗原决定族。利用此特性，探索了建立双单克隆抗体夹心法检测临床标本中ＩＬ－１β含量的可行性。     

抗人淋巴细胞单克隆抗体的化学发光标记及其在淋巴细胞CD3抗原检测中的应用

运用过碘酸钠做为交联剂将HRP表记到抗人淋巴细胞CD3抗原簇单克隆抗体上，其方法简便、快速、交联效果好。[结果]运用鲁米诺-过氧化氢体系，以新鲜人血中分离的淋巴细胞为抗原与标记抗体反应，测量时标记抗体最适稀释度为l：1000，最适pH为8．O，最适温度为25℃～30℃。     

纯化和标记抗人轻链β2m的单克隆抗体在细胞表面HLA-Ⅰ类分子检测中的应用

HLA-Ⅰ类分子在单个核细胞(PBMC)表面表达丰富,HLA-Ⅰ类分子除了参与全身免疫应答外,还有调节免疫和参与免疫细胞分化的作用.研究表明,PBMC的HLA-Ⅰ类分子在细胞免疫中发挥着极为重要的作用,其表达降低可能会减弱机体对肿瘤细胞的杀伤力,从而导致肿瘤细胞逃避机体免疫监视[1].     

抗极低密度脂蛋白受体单克隆抗体的大量制备及纯化

背景：极低密度脂蛋白受体被认为是一种＂瑞士军刀＂样多功能受体,对该受体的研究很广泛,但其商品化抗体种类较少,价格偏贵。目的：获得研究可用的抗极低密度脂蛋白受体的单克隆抗体。方法：为了获得抗极低密度脂蛋白受体单克隆抗体,选用可分泌针对极低密度脂蛋白受体胞内段的单克隆抗体的杂交瘤细胞IgG 6A6。通过将杂交瘤细胞IgG 6A6注入Balb/c小鼠腹腔内,2周后收集腹水,分别通过盐析法（正辛酸-饱和硫酸铵沉淀法）初步纯化和亲和层析进一步纯化目的蛋白,获得针对极低密度脂蛋白受体胞内段的单克隆抗体。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测其相对分子质量和纯度,酶联免疫吸附实验检测其活性。结果与结论：未纯化腹水Western Blotting结果显示条带较多,背景不干净;而经正辛酸-饱和硫酸铵法初步纯化的腹水、经Protein A进一步纯化得到的抗体均可特异识别极低密度脂蛋白受体。提示经盐析法和Protein A Agarose纯化可获得较纯和有活性的单克隆抗体。     

柯萨奇病毒A16型快速纯化和中和性单克隆抗体制备与鉴定

目的建立柯萨奇病毒A16型快速纯化方法,制备中和性单抗,并对单抗进行分析。方法收获CA16培养上清液,超滤浓缩,氯化铯密度梯度离心纯化病毒颗粒,透射电镜鉴定纯化产物。福尔马林灭活CA16,免疫BALB/c小鼠,制备分泌抗CA16特异性单抗的杂交瘤细胞系,用ELISA和中和试验分别对单抗特性进行分析。结果初步建立CA16病毒氯化铯密度梯度纯化方法,电镜显示,病毒颗粒为二十面体立体对称球形结构,病毒直径在20~30nm间,大小均匀。获得2株分泌抗CA16单抗的杂交瘤细胞系,2株单抗均为IgG2a亚型,Anti/CA16/5效价为103,Anti/CA16/10效价为104。2株抗体的中和效价分别为1∶256和1∶1 024。结论初步建立氯化铯密度梯度纯化CA16的方法,筛选出2株具有中和活性的抗CA16单抗,为CA16病毒的基础研究提供重要的原材料。     

抗人膀胱癌单克隆抗体BDI-1的扩增和鉴定

目的制备纯化的抗人膀胱癌单克隆抗体BDI-1。方法给Balb/c小鼠腹腔注射BDI-1杂交瘤细胞。收集的腹水过Pmtein G-sgsros亲和层析柱即得到人膀胱癌BDI-1单克隆抗体。采用ELISA法、间接免疫荧光法、SDS-聚丙烯酰胺凝胶电泳法和点印迹法鉴定单克隆抗体。结果每毫升腹水可以得到1-1．5mg的高效价（106）的纯化抗体。结论该方法可以扩增高效价的单克隆抗体．     

两株CD45类单克隆抗体的产生及其特性

CD45即白细胞共同抗原,它是由同一结构基因经不同拼接后形成分子量不同的一组蛋白产物,至少包括220、205、190和180kDa的四种肽链,因而CD45尚有CD45RO、CD45RA和CD45RB的亚美。CD45具有受体蛋白的结构特征,其细胞内部分为酪氨酸磷酸酯酶,参与白细胞的激活、分化和生长调控。本实验旨在研制CD45类的单克隆抗体,为相应的基础和应用研究提供技术手段。应用杂交瘤技术共获得两个均为小鼠IgG1亚类的单克隆抗体,分别命名为NN26-2和LN2-2,它们与正常人外周血、骨髓单个核细胞、胸腺细胞和多种人白血病细胞系的反应性与CD45的分布相一致。免疫沉淀的结果表明NN26—2识别分子量为180～220kDa的蛋白成分,计算机软件分析也证明NN26-2和LN2-2同属CD45类抗体。本文报道的两个单克隆抗体已被第五届国际人白细胞分化抗原研讨会所接受,可以用于肿瘤组织免疫病理鉴别诊断、恶性淋巴瘤免疫分型、淋巴细胞亚群和造血干细胞分离。     

人H-FABP单克隆抗体制备和纯化及其特性鉴定

目的制备人心肌型脂肪酸结合蛋白(Heart type fatty acid-binding protein;H-FABP)单克隆抗体。方法(1)应用淋巴细胞杂交瘤技术,将重组人H-FABP腹腔注射免疫BALB/C小鼠。(2)将鼠脾细胞与鼠骨髓瘤细胞(SP2/0)融合、培养。通过HAT和间接ELISA法筛选、鉴定制备稳定分泌抗人H-FABP单克隆抗体的杂交瘤细胞株。(3)利用Protein G Sepharose 4 Fast Flow亲合纯化体内诱生法制备的腹水抗体,并对抗体特性进行鉴定。结果得到7株特异性识别人H-FABP单克隆抗体,并纯化两株亲合力高、空间位点远的单克隆抗体。结论成功制备了稳定分泌抗人H-FABP单克隆抗体的杂交瘤细胞株。     

单倍体相合骨髓移植中急性移植物抗宿主病预防新方案的临床研究

目的　探讨骨髓移植 (BMT)供者应用G CSF和受者接受连续免疫抑制剂及加CD2 5单克隆抗体 (单抗 )预防急性重度移植物抗宿主病 (GVHD)的单倍体移植方案的可行性。方法　 38例白血病患者接受单倍体相合未去T细胞BMT ,供者应用G CSF动员 7d后采集骨髓 ;受者应用连续免疫抑制剂预防急性GVHD ,临床观察移植结果。结果　所有患者均取得三系造血重建 ,移植后粒细胞 >0 .5×1 0 9 L及血小板 >2 0× 1 0 9 L的中位时间分别为第 1 9天和 2 3天 ,植入直接证据检测证实完全供者造血 ,发生Ⅱ～Ⅳ度急性GVHD 4例 ,发生率为 1 0 .5 % ,无病存活大于 6个月可评价慢性GVHD 2 8例 ,均发生慢性GVHD ,表现轻微和局限 ,其中广泛性慢性GVHD 3例 ,发生率为 1 0 .7%。免疫功能恢复的结果显示 ,在移植后 1 8个月内动态观察免疫细胞计数的变化 ,CD3+、CD8+、CD1 9+、CD56+细胞在 1 2个月内可恢复至正常水平 ,CD4+细胞在移植后 1 8个月内恢复。随访中位时间 1 2个月 (6～ 2 6个月 ) ,死亡 1 2例 ,Kaplan Meier生存分析 ,2年估计无病生存率为 (68.4± 6 .0 ) % ,存活病例Karnofsky评分大于 90 %。结论　经G CSF动员的骨髓与连续免疫抑制剂的应用 ,特别是CD2 5单抗应用于未去T细胞单倍体相合BMT急性重度GVHD的预防 ,对移植物植入、免疫     

Beta 2-microglobulin determined by radioimmunoassay with monoclonal antibody

In this sensitive radioimmunoassay for beta 2-microglobulin (beta 2-M) involving a commercially available monoclonal antibody, we used a second monoclonal antibody, produced in our laboratory, for affinity-chromatographic purification of beta 2-M. Both monoclonal antibodies bound to all of the charge isomers of beta 2-M identified by two-dimensional gel electrophoresis. Polyethylene glycol was used to separate the phases after incubation for 3 h at room temperature. Sensitivity was 0.25 ng of beta 2-M. Within-assay CVs were less than 5.4%, between-assay CVs less than 7.3%. Analytical recovery was 95-102%. The reference interval was 1.2-2.2 mg/L for 49 healthy subjects. The correlation coefficient for comparison with a polyclonal antibody assay was 0.997 for 44 patients. In a study correlating serum beta 2-M and creatinine concentrations with glomerular filtration rate for 50 patients, correlation coefficients for log-log transformed data were -0.94 and- 0.95, respectively. Concentrations of beta 2-M were increased in serum of patients with imparied renal function and also in patients with normal renal function who had disorders involving the immunologic response. We found no clear-cut advantage of measuring serum beta 2-M over serum creatinine in the estimation of glomerular filtration rate.     

Double monoclonal time-resolved immunofluorometric assay of beta 2-microglobulin in serum

Previously reported immunochemical assays of beta 2-microglobulin (beta 2m) have usually been based on polyclonal antisera. We have developed a "sandwich"-type time-resolved immunofluorometric assay (TR-IFMA) for beta 2m in serum, based on two monoclonal antibodies against human beta 2m. Microtiter wells are coated with the capture antibody, and the tracer antibody is labeled with a europium chelate. In a simple and fast assay procedure, prediluted serum samples are incubated with the tracer for 1 h in the microtiter wells, after which the wells are washed and the fluorescence of Eu is measured. The mean analytical recovery was 101.8% and results by TR-IFMA showed a good linear correlation with those by an established radioimmunoassay. The analytical range of TR-IFMA is large and well suited for clinical purposes.     

Radioimmunoassay of beta2-microglobulin.

A radioimmunoassay for quantitation of beta2-microglobulin in serum and urine is described. Polyethylene glycol 6000 was used to precipitate the antigen-antibody complex. The assay can be performed within 5 hr. In healthy human subjects (mean age, 30.6 years and 42.0 years for serum and urine determinations, respectively) the mean concentration of beta2-microglobulin in serum was 125 nmol/l (range, 49-190 nmol/l). In men the mean 24-hr urinary excretion was 12.9 nmol (range, 8.6-19.2 nmol), and in women it was 5.5 nmol (range, 2.9-8.7 nmol). The assay range was 0.23-19.40 nmol/l, and the detection limit was 0.042 nmol/l, using a prolonged incubation time. The coefficient of variation based on 20 determinations in serum dilutions with a concentration of beta2-microglobulin of 8.14 and 1.92 nmol/l was 8.1 per cent and 9.0 per cent, respectively.     

Urinary excretion of albumin beta2-microglobulin and free light chains during lithium treatment.

Albumin, beta2-microglobulin and free light chains were determined in urine in nine manic-depressive patients before and at intervals during three months of lithium treatment (longitudinal study). The same determinations were carried out in twenty-seven manic-depressive patients who had been treated with lithium for 3 months to 20 years and also in a control group (transversal study). There were no statistically significant changes in urinary excretions of albumin, beta2-microglobulin and free light chains during the longitudinal study. In one patient albumin excretion gradually increased during the study and remained elevated on reexamination 1 year later. No significant differences were found between the lithium treated patients and control subjects in the transversal study in either albumin, beta2-microglobulin or free light chain excretion. It is not clear whether the increased and sustained albumin excretion in one of the patients was due to lithium or was conincidental. The study shows that in most patients lithium treatment does not affect renal protein excretion.     

Pregnancy-specific beta 1-glycoprotein in serum in monoclonal gammopathies: relationship with serum beta 2-microglobulin, and cellular origin

Pregnancy-specific beta 1-glycoprotein (SP1) was assayed by particle-counting immunoassay in serum from 86 healthy blood donors and 236 patients with various types of gammopathy. A concentration of 1 microgram/L was taken as the upper normal limit. Abnormally high values were found in one of 10 patients with monoclonal gammopathy of undetermined significance, in 65% of 152 patients with multiple myeloma, in 84% of 64 patients with Waldenstr?m's macroglobulinemia, and in seven of 10 patients with monoclonal gammopathies associated with other myeloproliferative disorders. In a study of 90 myeloma patients, the SP1 value correlated (p less than 0.001) with the concentration of beta 2-microglobulin in serum, a value which had been corrected for possible renal dysfunction, but not with the concentration of the monoclonal component. SP1 was detected by direct immunofluorescence in myeloma cells of bone-marrow smears from six of 10 patients with myelomatosis. These six patients had serum SP1 values greater than 1 microgram/L, whereas the four patients with fluorescence-negative myeloma cells had SP1 values less than 1 microgram/L.     

抗人TIM-3单克隆抗体的纯化及其体外生物学功能的研究

目的纯化抗人T细胞免疫球蛋白粘蛋白3(TIM-3)单克隆抗体4E8,检测TIM-3单克隆抗体4E8的体外生物学功能。方法利用单克隆抗体纯化技术获取4E8;用细胞转染技术获得表达人TIM-3分子的细胞系,流式细胞术检测4E8与人TIM-3分子结合的能力以及阻断人TIM-3融合蛋白(TIM-3 Ig-hu Fc)与凋亡细胞表面TIM-3配体磷脂酰丝氨酸(Ptd Ser)结合的能力;混合淋巴细胞反应(MLR)及ELISA法检测4E8对CD4+T细胞分泌IFN-γ的影响。结果 4E8可与人TIM-3分子结合,但不可阻断TIM-3与Ptd Ser的结合。与阴性对照组(958.3±153.2)相比,4E8(10μg/m L组:2 563±150.3,3.33μg/m L组:1 981±211.5)可增强MLR实验中CD4+T细胞分泌IFN-γ的能力,差异有统计学意义(P0.05)。并且与单独使用4E8(10μg/m L组:1 981±211.5,0.33μg/m L组:844±76.2)相比,4E8与程序性死亡受体1(PD-1)抗体nivolumab联合使用(10μg/m L组:3 049±80.5,0.33μg/m L组:1 957±321.3)有协同作用,差异有统计学意义(P0.05)。结论成功纯化出抗人TIM-3单克隆抗体4E8。4E8具有增强CD4+T细胞分泌IFN-γ的能力,但对CD4+T细胞的作用不依赖于阻断TIM-3与其配体Ptd Ser的结合。     

Serum beta 2-microglobulin in hepato-biliary diseases

The levels of serum beta 2-m in hepato-biliary disease have been examined. Significant increase has been observed in various benign and malignant diseases of the liver, but not in jaundice due to acute pancreatitis. The age-dependent relationship of beta 2-m is present in controls, weaker in the malignant diseases and absent in the benign diseases. The level of beta 2-m is not influenced by jaundice. Serum beta 2-m levels have no role as a discriminant in liver disease.