抗人白细胞间素-2 McAb亲和力排列及亲和指数的测定

利用蛋白浓度校正的ELISA稀释曲线快速确定了抗人白细胞间素-2(IL-2)单克隆抗体(McAb)的亲和力排列。该排列与用竞争ELISA测定的亲和常数(K_D值)相一致。为了更实际地确定McAb的用途,又用十二烷基磺酸钠(SDS)等解离因素,建立了反映 McAb在实际应用时亲和力变化强度的吸附-解离ELISA,测定McAb的亲和指数(ID)。根据亲和力排列和亲和指数可更准确地取舍McAb,供检测或亲和色谱使用。     

抗人白细胞间素-2多克隆及单克隆抗体的生物学特征

利用人工合成的白细胞间素2(IL-2)多肽片段,用中和试验、竞争ELISA和染色IL-2分泌细胞的碱性磷酸酶抗碱性磷酸酶桥联酶标技术,对己制备的抗人IL-2多克隆抗体及单克隆抗体(McAb)的表位特异性、中和活性、亲和常数及杭天然IL-2的反应性进行了全面鉴定。结果表明:利用多种免疫方案、融合前强化加强免疫及多个脾脏混合融合制备策略所获得的McAb,在生物学特征上表现出多样化,即这些McAb的表位特异性、中和活性、亲和力及抗天然IL-2活性各不相同。其中抗IL-2 C末端的McAb为首次报道。这组McAb适用于对IL-2研究的不同目的。     

抗人μ链单克隆抗体特性鉴定及临床初步应用

<正> IgM是血清中重要的免疫球蛋白之一,其含量测定与许多疾病的诊断有密切关系。我们从巨球蛋白血症患者血清中纯化了IgM作为抗原免疫BALB/c小鼠,用细胞融合技术获得了分泌抗入μ链单克隆抗体(McAb)杂交瘤细胞株,并对此McAb特性进行鉴定及临床初步应用。 一、特性鉴定: (一)所获二个细胞株(2D_6、E_4)的染色体分别     

人IL-2抗独特型单克隆抗体

本文报道吸附纯化兔抗人重组IL-2抗体(Id)的抗独特型单克隆抗体(抗-IdMcAb)6A12。McAb与Id结合可以抑制Id对IL—2的中和作用,表明其结合位点可能与Id上IL—2中和位点相同或邻近。6A12协同IL—2对PHA活化人T细胞的增殖作用,纯化的6A12 IgG也可与纯化的重组IL-2受体可溶性P~(55)亚单以结合;对IL-2依赖的鼠T细胞系(C TLL),纯化的6A 12IgG有抑制IL-2爱性作用,而当IL-2过量时可使抑制作用逆转。结果表明:6A12不可能是IL-2受体结合位点精确的“内都影像”,但它至少可与高亲和力IL-2受体复合物中配体结合位点P~(55)亚单位结合。特定配体的抗-Id抗体与配体的生理受体相互作用已有系统描述,对人重组白细胞干扰素A(rIFN-αA )抗体的抗-Id McAb研究已知:McAb 3B1似乎完全可以作为干扰素内部影像或Jerne称作的“补位诱导抗体(Ab2β)”。T淋巴细胞表面具有对IL-2亲和力高低不同的膜受体,IL-2与之结合调节T细胞生长。最近有关受体结构研究也表明。高亲和力IL-2受体复合物至少由2种多肽链构成,α链(Tac抗原或P~(55))及β链(70～75kD糖蛋白或P70),二者分别以低等(P~(55))和中等亲和力独立与IL-2结合。本文报道研制了人IL-2抗-Id MeAb 6A12,并对其生物学活性进行了研究。     

B细胞增殖分化因子(TRF/IL-5)及其受体

已知作用于B细胞的淋巴因子有IL-1、IL-2、B细胞刺激因子-1(BSF-1)、IL-4、T细胞替代因子(TRF)/IL-5及B细胞刺激因子-2(BSF-2)等。作者在可溶性因子TRF的研究中,纯化了TRF,制成了抗TRF的McAb,同时完成了     

单克隆抗体ELISA双抗体夹心法测定人白细胞介素2

本文叙述了测定人白细胞介素2(IL-2)含量的ELISA双抗体夹心法。用辣根过氧化物酶标记IL-2单克隆抗体。聚苯乙烯反应板预先用0.1％戊二醛处理,然后以单克隆抗体包被。本法批内变异系数CV=3.99％,批间变异系数CV=5.96％,标准曲线的测定范围为3.1～100u/ml,检测下限为1.55u/ml。应用本法已测定了2S例正常人和11例恶性肿瘤患者的IL-2含量。     

抗人白细胞间素-2单克隆抗体的生物学特征及其应用

采用多途径、高效价免疫及强化加强免疫方案,制备了一组生物学特性多样化的抗人自细胞间素-2(IL-2)的单克隆抗体(McAb)。利用人工合成的IL-2多肽片段,夹心排阻ELISA亲和指数测定及中和试验等方法,全面鉴定了这些McAb,利用     

一种检测可溶性白细胞介素2受体夹心法ELISA的建立

采用两种识别不同表位的小鼠抗人白细胞介素2受体(IL-2R)单克隆抗体(McAb),在国内首次建立了检测可溶性IL-2R(sIL-2R)的夹心法ELISA,并进行了初步应用。结果显示:本法特异性强、敏感性高、重复性好、操作简便,能检出正常人、某些患者血清中以及细胞培养上清中sIL-2R,可用于基础和临床免疫学研究。     

一个简单敏感的检测IL-2活性的生物学方法

本文介绍了一种简单一次定量的检测IL-2生物活性的方法。它以培养纯CD3~+T细胞为基础,CD3~+T 细胞是从血液的单个核细胞中,用微球(M450)结合CD3McAb 筛选的。培养时,经抗CD3活化的T 细胞将表达IL-2受体而不产生IL-2。加入IL-2后,可引起增殖反应,IL-2浓度在0.01～20U/ml时,可以得到一个线性剂量-反应曲线。细胞对IL-1、IL-4、TNF-α、IFN-α、INF-γ、PHA、ConA 不反应。IL-2的反应性可为TNF-α增强,被IFN-α抑制。试验用的促分裂素不影响增殖反应。抗IL-2和抗IL-2受体的抗体可以抑制IL-2反应,并有一定的剂量依赖关系。本法既简单又特异,不用长期培养实验用的细胞。     

抗人IL-5单抗的制备、特性及其在ELISA中的应用(文摘)

IL-5是一种对于不同的靶细胞具有不同免疫调节活性的细胞因子,它在B细胞以及其他造血细胞(如T细胞和嗜酸性粒细胞)的生长和分化过程中起着重要的作用。Harada(1987)和Schumacher等(1988)曾报道抗小鼠IL-5的大鼠单克隆抗体(McAb)与人IL-5有交叉反应性。本文报告抗人IL-5的小鼠McAb的制备。这种小鼠McAb可用于免     

Improvements in the measurement of stool decay-accelerating factor in the detection of colorectal cancer

We have previously developed an enzyme-linked immunosorbent assay (ELISA) to measure stool decay-accelerating factor (DAF) and found that stool DAF concentrations were significantly elevated in patients with colorectal cancer, suggesting that the measurement of stool DAF may be valuable for the detection of colorectal cancer. In order to refine the assay for the measurement of stool DAF, we investigated 1) effects of centrifugation of stool samples, 2) effects of detergents, and 3) adequate combination of various anti-DAF monoclonal antibodies for the ELISA system using only monoclonal antibodies. We found that high-speed centrifugation could be omitted and that only the removal of large undigested food residues by centrifugation of short duration in a low-speed benchtop microcentrifuge sufficed to adequately prepare the stool samples. Addition of 2 detergents, octyl beta-glucoside and sodium deoxycholate, known to solubilize glycosyl-phosphatidylinositol-anchored proteins such as DAF, did not influence stool DAF values. By using 2 mouse anti-DAF monoclonal antibodies (clone 4F11 and 1C6), we were able to achieve a stable ELISA for the measurement of stool DAF using a uniform source of antibodies. The results should allow us to consistently apply the DAF assay for routine use in the detection of colorectal cancer.     

Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis.

Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 10(3) organisms, whereas FT2F11 was able to detect only 10(5) organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified O side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the O side chain and that FT2F11 is directed against the core.     

Production and characterization of monoclonal antibodies directed against Bordetella pertussis lipopolysaccharide.

Hybrid cell lines producing monoclonal antibodies against Bordetella pertussis lipopolysaccharide (LPS) were established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and ELISA-inhibition experiments with LPS and delipidated polysaccharide fragments (PS-1 and PS-2) prepared from B. pertussis LPS. Monoclonal antibody 9-1-H5 reacted with B. pertussis LPS only, whereas monoclonal antibodies 6-4-H6 and 9-2-A8 reacted with PS-1 and PS-2 as well as B. pertussis LPS. The antibodies did not react with LPS prepared from B. parapertussis and B. bronchiseptica in an LPS-specific ELISA. A monoclonal antibody-based sandwich ELISA was developed for detection of B. pertussis LPS. This assay had a detection limit of B. pertussis LPS in concentrations ranging from 0.16 to 0.32 microgram/ml. The assay was also shown to be specific for the detection of whole B. pertussis bacteria. No cross-reactions were observed with strains of Branhamella catarrhalis, Neisseria meningitidis, Streptococcus miteor, Haemophilus influenzae, or Legionella pneumophila. The monoclonal antibodies might be useful for the detection of soluble antigens and whole bacteria in clinical samples and for studies of the immunochemical structure of B. pertussis LPS.     

Production, characterisation and use of monoclonal antibodies to human interleukin-5 in an enzyme-linked immunosorbent assay

Two mouse monoclonal antibodies (Mabs) against recombinant human interleukin-5(rhIL-5) have been produced, characterised and purified. Both are IgG1 antibodies and neutralised the activity of rhIL-5 in the B13 assay. Neither Mab cross-reacted with mouse IL-5. A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was developed with different combinations of the mouse Mabs and also a rat anti-mouse IL-5 Mab, TRFK5, which also has activity against rhIL-5. The most sensitive assay, with a lower detection limit of 0.5 ng/ml IL-5, used TRFK5 as the capture antibody and the mouse anti-human IL-5 Mab as second antibody. The sensitivity of this assay was increased by an enhanced chemiluminescent reagent and resulted in a lower limit of detection around 40 pg/ml IL-5.     

诺如病毒表达衣壳蛋白单克隆抗体的制备、鉴定和初步应用

目的:建立能稳定分泌抗诺如病毒(Norovirus)N蛋白的单克隆抗体(mAb)细胞株, 制备抗诺如病毒核衣壳蛋白的mAb, 为诺如病毒的早期快速检测及致病机制的研究提供实验材料.方法:用E.coli表达的GⅡ组广州株NVgz01(DQ369797)Norovirus-N蛋白免疫BALB/c小鼠, 通过PEG使小鼠脾细胞和Sp2/0细胞融合, 使用HAT选择性培养基培养融合细胞, 用间接ELISA和Western blot测定mAb的效价、免疫球蛋白亚型和mAb的特异性, 并将各mAb之间进行配对.结果:通过细胞融合和克隆化, 共筛选出4株分泌抗Norovirus-N蛋白抗体的杂交瘤细胞株N2C3、 N7C2、 N4B1、 N8A9.间接ELISA和Western blot检测结果表明, 这4株mAb都可以与E.coli表达的GⅡ组广州株Norovirus-N蛋白产生特异性反应, 并且能与天然粪便标本中的GⅡ组Norovirus产生特异性反应.配对结果显示N2C3和N7C2之间配对, 对表达蛋白和天然病毒都具有较强的检测灵敏度.结论:获得了诺如病毒GⅡ组特异性mAb, 为制备免疫诊断试剂盒及致病机制的研究奠定了基础.     

抗人肝再生增强因子单克隆抗体的研制

目的　利用纯化的重组人肝再生增强因子(hALR)制备抗hALR的单克隆抗体,建立hALR的检测方法。方法 采用杂交瘤技术制备单克隆抗体,经ELISA和免疫印迹证明其特异性。结果获得了4株分泌抗hALR特异性抗体的杂交瘤 细胞系;无血清培养液效价为1×10-2,腹水效价为1×10-5;亚类鉴定表明2株单克隆抗体为IgGl,另2株为IgG2b;免疫印迹 显示抗体结合抗原的分子量与hALR相符,为特异性抗hALR抗体。结论通过杂交瘤技术,获得了抗hALR的单克隆抗体 为以后的工作奠定了基础。     

Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of Bordetella pertussis filamentous hemagglutinin.

Hybrid cell lines producing monoclonal antibodies against Bordetella pertussis filamentous hemagglutinin (FHA) were established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroblotting. The monoclonal antibody-based sandwich ELISA was developed for detection of B. pertussis FHA. The assay had a detection limit of B. pertussis FHA in concentrations ranging from 7 to 15 ng/ml. The assay was also able to detect whole B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica bacteria. No cross-reactions were observed with strains of Branhamella catarrhalis, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Streptococcus miteor, or Streptococcus pneumoniae. The monoclonal antibodies might be useful for the detection of soluble antigens and whole bacteria in clinical samples and for studies of the immunochemical structure of B. pertussis FHA.     

Monoclonal antibodies to guinea pig interferon-gamma: tools for cytokine detection and neutralization

We have generated polyclonal antisera and monoclonal antibodies against recombinant guinea pig IFN-gamma. These antibodies were used to inhibit the function of IFN-gamma in vitro and to establish a capture ELISA system for the detection and quantitation of this cytokine. Although recombinant protein expressed in E. coli was available in abundance, it was only of limited value to develop a capture ELISA which detects the native cytokine, since only a limited number of monoclonal antibodies reacted both with the recombinant and the native protein. Positive test results in an initial ELISA setup with recombinant IFN-gamma were not predictive for the detection of IFN-gamma from activated T-lymphocytes in the same assay. After evaluating several different combinations of rabbit antisera and monoclonal antibodies, an assay system was established which uses two mouse monoclonal antibodies as capture and detecting reagents. Three of the monoclonal antibodies and the rabbit antisera were able to block the function of guinea pig IFN-gamma when assayed in a luciferase reporter assay.     

阻断型CD154抗体和CD80抗体生物学作用的研究

为比较阻断型抗人CD154单克隆抗体(1B1)和抗人CD80单克隆抗体(3H8)的单独及联合应用在调节CD4+T细胞对同种抗原的初次和再次免疫应答中的作用。采用免疫磁珠阴性选择法分离获得人外周血CD4+T细胞、将纯化的CD4+T细胞与刺激细胞体外共培养,通过检测培养上清IL-2、IFN-γ水平以及CD4+T细胞的增殖来评价1B1和3H8的生物学作用。结果显示,1B1和3H8单独或联合应用能不同程度地抑制混合淋巴细胞反应中CD4+T细胞对同种抗原刺激的增殖,下调CD4+T细胞分泌IL-2和IFN-γ,并且在再次反应中能有效诱导CD4+T细胞对同种抗原的免疫低反应性。因此,1B1和3H8的单独及联合应用在移植排斥的免疫干预及同种抗原免疫耐受的诱导中具有潜在的应用前景。