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1.
间接ELISA法用于细胞抗原特异的单克隆抗体筛选 总被引:4,自引:0,他引:4
在单克隆抗体(mAb)制备过程中,间接ELISA方法用于可溶性蛋白为抗原的mAb筛选。当得不到可溶性抗原的情况下,用细胞膜抗原或克隆抗原分子,再以痘苗病毒为载体转入真核细胞制备mAb,可以用间接免疫荧光法进行筛选。由于此种方法费时,所需细胞数量大,影... 相似文献
2.
抗人白细胞单克隆抗体的特异性及其特性鉴定 总被引:1,自引:0,他引:1
目的制备抗人白细胞单克隆抗体(mAb),并对其识别抗原及组织的特异性进行鉴定。方法采用分离的人全白细胞悬液免疫Balb/c小鼠,取脾细胞与Sp2/0细胞常规融合,用间接ELISA筛选mAb,流式细胞仪及免疫组化染色SP法鉴定其组织特异性。 结果成功地制备1株抗人白细胞 mAb,命名为SZ-105。ELISA法测定其腹水效价为1×10-5。琼脂糖双扩散鉴定为IgG1类。流式细胞仪间接免疫荧光技术及免疫组化SP法测定表明,mAb SZ-105可选择性地与外周血液的粒细胞、单核细胞和淋巴细胞呈强阳性反应,而与红细胞及血小板不出现反应。该mAb还可与正常人骨髓的白细胞前体起反应。另外,在肝、肺、脾、胸腺及淋巴结正常组织中的巨噬细胞表面,也有SZ-105抗原表达。SZ-105抗原经亲和层析纯化,再经SDS-PAGE 和Western blot证实,mAb SZ-105识别的抗原是相对分子质量(Mr)为75 000左右的单链蛋白多肽。结论 获得1株具有高度免疫学活性和组织特异性的抗人白细胞mAb SZ-105,其对研究白细胞的分化、免疫功能,以及隐匿性急、慢性炎症疾病的放免显像诊断都具有一定的临床意义。 相似文献
3.
抗人肝癌单克隆抗体HCD78的制备及免疫组化定位 总被引:1,自引:0,他引:1
原发性肝癌是世界上最常见的恶性肿瘤之一,具有发病隐袭、病死率高的特点,早期诊断对预后的意义重大。单克隆抗体(mAb)技术问世后,人们试图通过应用抗肝癌mAb提高肝癌的早期诊断率。然而目前获得的抗肝癌mAb识别的抗原都不是肝癌特异性抗原,而且由于肿瘤异质性的存在,又使其临床应用受到一定影响。本研究应用自建的人肝癌细胞株HC108免疫小鼠,制备了1株能稳定分泌抗人肝癌mAb的杂交瘤细胞。免疫组化证实,该mAb具有较好的特异性,其识别的肿瘤抗原主要定位于细胞膜。现将建株过程和初步鉴定的结果报道如下:1… 相似文献
4.
Fas/Apo1/CD95抗原的发现,始于对两株单克隆抗体(mAb)的功能观察,即抗Apo1mAb和抗FasmAb〔1,2〕。由于二者都能识别细胞膜上CD95蛋白抗原,且能诱导敏感细胞凋亡,才导致今天对Fas抗原的生物医学研究。制备抗FasmAb对于Fas抗原的深入研究,尤其是对Fas抗原信号传导机制的阐明,以及生物医学应用都有重要的意义。本文利用Fas+的细胞作为免疫原,制备了1株鼠抗人FasmAb2A1。1材料和方法1.1材料1.1.1细胞系Sp2/0小鼠骨髓瘤细胞、Jurkat人T淋巴… 相似文献
5.
噬菌体呈现的随机肽库已成功地用于筛选B细胞或T细胞表位。为进一步研究胃癌相关抗原与相应单克隆抗体(mAb)结合的结构基序及其相互作用,我所通过两株胃癌mAbMG7和MGb2筛选噬菌体呈现的随机九肽库,获得一些可与mAb结合的噬菌体呈现肽。我们采用竞争抑制实验,研究了这些肽与天然胃癌相关抗原的关系。1材料和方法1.1材料胃癌mAbMG7和MGb2由本所研制〔1,2〕,通过筛选噬菌体呈现的随机九肽库〔3,4〕,获得可与相应mAb结合的噬菌体呈现肽〔5,6〕。1.2方法1.2.1噬菌体呈现肽竞争抑制试验将纯… 相似文献
6.
1985年, Burns等[1]以活化的 T细胞为免疫原,制备了抗活化T细胞表面抗原的单克隆抗体(mAb),并将其中 1株mAb Leo-A1识别的抗原命名为人T细胞谱系特异性活化抗原 1(T lin-eage-specific activation antigen1, TLiSA1)。 mAbLeo-A1在混合淋巴细胞反应中,可抑制细胞毒T细胞(cytotoxic T lymphocytes, CTL)分化。随后Scott等[2]发现, TLiSA1也高表达于血小板表面,可刺激血小板的活化和凝集,遂将此抗原… 相似文献
7.
A10、E5和F11是3株抗人肝细胞癌的单克隆抗体(mAb)。经ABC免疫组化染色法证实,这些mAb针对的抗原在8例肝癌组织中呈高表达,A10、E5和F11分别为6/8,5/8和6/8;而在肝炎中呈低表达,分别为2/11、0/11和1/11[1]。故这几株mAb所针对的相应抗原可能是肝癌相关抗原。作为筛选肝癌细胞表达文库的候选抗体,通过Western免疫印迹反应可了解其相应抗原的一些性质及确定mAb对变性抗原的识别能力。1 材料和方法1-1 细胞株和mAb 肝癌细胞株SMMC7721与HHCC… 相似文献
8.
抗B-CLL Id x抗CD3双特异性抗体的制备及其特性研究 总被引:1,自引:0,他引:1
应用无筛选标记细胞株杂交瘤·杂交瘤细胞直接融合法制备了2株分泌抗慢性B淋巴细胞白血病(B-CLL)Idx抗CD3双特异性抗体(BsAb)的四价体瘤细胞株。采用间接ELISA试验与间接免疫荧光试验证明了该BsAb能分别与B-CLL患者血清及带CD3标记的细胞特异性结合,经细胞结合试验证明该BsAb同时具备与Id及CD3两种抗原结合能力,经酶桥联法证明该BsAb亚类为IgG1×IgG2a,用MTT法证实BsAb能诱导T细胞活化增殖 相似文献
9.
用重组牛bFGF免疫Balb/c小鼠,通过细胞融合,以反向间接血凝和间接ELISA筛选,以及有限稀释法克隆化,建立了9株稳定分泌抗bFGF单克隆抗体(mAb)杂交瘤细胞。对其中3株用Westernblot和生物活性抗体中和实验进行了鉴定。结果显示,mAb可结合重组牛或人bFGF,并能抑制bFGF刺激Balb/c3T3细胞的生长;腹水的ELISA滴度为1∶3000;用其中2株mAb建立的双抗体夹心ELISA法灵敏度可达50pg/孔。本文还讨论了抗bFGFmAb的应用价值。 相似文献
10.
鼻咽癌抗独特型抗体诱导的体液和细胞免疫应答 总被引:5,自引:0,他引:5
目的 探讨抗独特抗体2H4和5D3模拟抗原诱导免疫应答的能力。方法 用Ab2免疫Balb/c小鼠获得抗血清,以ELISA检测其Ab3,Western Blot分析其识别抗原的分子质量。用迟发型超敏反应(DTH)和淋巴细胞增殖试验,检测Ab2诱发细胞免疫反应的能力。结果 2H4,5D3免疫同系动物产生的含有抗抗蚀特型抗体(Ab3)的抗血清,可特异性地与靶细胞结合。FC2(Abl)和经2H4免疫的Ab 相似文献
11.
The functional valency of a monoclonal antibody (mAb) has important influences on such things as antigen avidity, Fc-mediated immune effector functions, and clearance of immune complexes. cV1q, a neutralizing rat/mouse chimeric anti-mouse tumor necrosis factor (TNF) monoclonal antibody (mAb), and Rt108, a neutralizing mouse anti-rat TNF (anti-raTNF) mAb, appear to be functionally monovalent for TNF-binding despite containing two antigen binding sites. The functional monovalency of these two independent anti-rodent TNF mAbs is presumably a result of steric hindrance from one TNF molecule binding to one Fab arm that prevents binding of a second TNF molecule to the other Fab arm. To test whether this steric hindrance could be overcome by introducing extra space and flexibility between the Fab arms, these mAbs were engineered to contain an extra CH1 immunoglobulin domain between the CH1 and hinge domains of their heavy chains. In vitro binding data showed that, compared to the original mAbs, the modified mAbs (S-mAbs) had greater capability of binding two TNF molecules simultaneously. In vitro activity assays showed that, compared to the original mAbs, the S-mAbs had significantly greater TNF-neutralization potency, with the S-mAb version of cV1q (S-cV1q) being 200-fold more effective at blocking mouse TNF (muTNF) and the S-mAb version of Rt108 (S-Rt108) being 20-fold more effective at blocking raTNF. Similar results were observed in vivo, where S-cV1q was between 100- and 500-fold more protective than cV1q in mice challenged with endotoxin. These data reveal that introduction of another constant region immunoglobulin domain into two unrelated mAbs dramatically enhanced their neutralization potency. Other mAbs may also show more potent activity using this engineering approach, particularly mAbs that recognize homopolymeric antigens. 相似文献
12.
Generation of monoclonal antibodies to coelomocytes of the purple sea urchin Arbacia punctulata: characterization and phenotyping 总被引:1,自引:0,他引:1
Cellular cytotoxicity is a key component of animal innate immune responses that is one of the first lines of defense against invaders. There is increasing interest in the study of the cellular immune response, particularly non-specific cytotoxic cells and natural killer cells and their receptors. Studies of non-specific cytotoxic cell and natural killer cell recognition and killing (and the receptors involved) will reveal new and important insights into cellular mechanisms of host defense. Here we describe mAbs specific for coelomocyte sub-populations of the purple sea urchin, Arbacia punctulata, using highly purified coelomocyte populations as the antigen source. Monoclonal antibodies were selected using flow cytometric screening methods. Several of the mAbs were shown to bind to two sub-types of coelomocytes when assayed by fluorescence microscopy. Furthermore, these mAbs inhibited coelomocyte cytotoxicity against vertebrate target cells in a functional assay. The mAbs have been used in immunoprecipitation studies. 相似文献
13.
Human T-cell leukemia-associated cell surface glycoprotein GP37: studies with three monoclonal antibodies and a rabbit antiserum 总被引:2,自引:0,他引:2
B K Seon T Fukukawa A L Jackson D Chervinsky C K Tebbi A I Freeman H Matsuzaki 《Molecular immunology》1986,23(6):569-580
Three monoclonal antibodies (mAbs), termed SN2, SN2a and SN2b, were used in the present work to study a human T-cell leukemia-associated cell surface glycoprotein, GP37. Strong specificity of mAbs SN2, SN2a and SN2b for T leukemia cells was demonstrated by radioimmunoassay and fluorescence-activated cell sorter (FACS) analysis. GP37 was not detected on normal human peripheral blood lymphocytes, purified normal T-cells, normal thymocytes nor normal bone marrow cells. Furthermore, GP37 was barely detectable on phytohemagglutinin (PHA)- and Concanavalin A (Con A)-activated T-cells. The results indicate clinical utility of these mAbs. Competitive binding experiments show that the epitopes recognized by SN2 and SN2a are sufficiently close to each other to allow complete reciprocal inhibition of binding whereas the epitopes recognized by SN2 and SN2b are less close to allow only partial reciprocal binding inhibition. The biochemical nature of antigenic determinants defined by these mAbs was studied by treating T leukemia cells with trypsin, chymotrypsin, thermolysin, neuraminidase and mixed glycosidases. The results suggest that the antigenic determinants defined by these mAbs all consist of the protein moiety of the glycoprotein GP37. No significant antigenic modulation was observed when T leukemia cells were reacted with SN2. In a sequential immunoprecipitation experiment, a 125I-labeled leukemia antigen preparation was first treated with a rabbit anti-T leukemia antiserum. The latter had been prepared by immunizing a rabbit with a partially purified human T leukemia antigen preparation and showed a good specificity for T leukemia cells. Subsequent treatment of the labeled antigen preparation with SN2 showed that SN2 antigen had been precleared. Thus, both mouse mAb SN2 and the rabbit anti-T leukemia antiserum react with the same GP37 molecule. 相似文献
14.
CD147 monoclonal antibodies induce homotypic cell aggregation of monocytic cell line U937 via LFA-1/ICAM-1 pathway 总被引:11,自引:0,他引:11
CD147 is a 50 000-60 000 MW glycoprotein of the immunoglobulin superfamily broadly expressed on haemopoietic cell lines and peripheral blood cells. In the present study, six monoclonal antibodies (mAbs) directed against the CD147 protein were generated. The antigen defined by the generated CD147 mAbs is widely expressed on haemopoietic cell lines, peripheral blood cells and is a lymphocyte activation-associated cell surface molecule. The generated CD147 mAbs precipitated a broad protein band from U937 cells of 45 000-65 000 MW under reducing conditions. Functional analysis indicated that the CD147 mAbs markedly induced homotypic cell aggregation of U937 cells, but not K562 cells. The CD147 mAb-induced cell aggregation was inhibited by leucocyte function-antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) mAbs. However, the expression of LFA-1 and ICAM-1 molecules on U937 was not altered by CD147 mAb treatment. The U937 cell aggregation induced by CD147 mAb was also inhibited by ethylenediamine tetra-acetic acid (EDTA), sodium azide and when incubated at 4 degrees. We therefore propose that the binding of CD147 mAb to CD147 molecule, which mimics the natural ligand binding, may generate intracellular signals that activate LFA-1/ICAM-1 intercellular adhesion pathway. 相似文献
15.
抗HIV-1核心抗原p24单克隆抗体的制备及特性的初步鉴定 总被引:3,自引:0,他引:3
目的 :建立分泌抗HIV 1p2 4单克隆抗体 (mAb)的杂交瘤细胞株 ,并对其特性进行初步鉴定。方法 :以纯化的基因工程制备的p2 4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HAT、HT选择培养及有限稀释法进行克隆化后 ,用间接ELISA法及Dotblot对其进行筛选和特性鉴定。结果 :筛选到 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,其腹水效价为 1×10 -5,亲和力为 1.7× 10 4~ 1.8×10 4mol/L ,mAb的Ig亚类均为IgG1。两株mAb与HBcAg、HCVRNA阳性血清及HIVgp4 1等均无交叉反应 ,只与HIV 1p2 4抗原阳性血清产生特异反应。结论 :成功地建立了 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,为进一步研制HIV 1p2 4抗原的ELISA检测试剂盒奠定了基础 相似文献
16.
Recognition sites of two monoclonal antibodies (mAbs), 1B7 and 1E4, specific for murine MHC class II were determined, using eleven L cell transfectants which express recombinant class II (I-A) molecules. 1B7 positive cells possessed a haplotype k in the α-helix of the β-chain, whereas 1E4 positive cells had haplotype b at the α-helix of the β-chain. The α-helix of the β-chains is regarded as an important site for antigen binding. Thus, the 1B7 and 1E4 mAbs may be useful for analysis of functional sites on class II molecules involved in antigen recognition. 相似文献
17.
CD6 is a type I T‐cell surface receptor that modulates antigen receptor signalling. Its activity is regulated by binding of its membrane proximal domain (domain 3) to a cell surface ligand, CD166. CD6 monoclonal antibodies (mAbs) specific for the membrane distal domain (domain 1) perturb CD6 function including itolizumab (Alzumab?), which has reached the clinic for treatment of autoimmune disease. We characterized molecular and functional properties of several CD6 mAbs including itolizumab to define potential mechanisms of action. Epitope mapping using the crystal structure of CD6 to design mutants identified two distinct binding sites on different faces of domain 1, one containing residue R77, crucial for MT605 and T12.1 binding and the other, E63, which is crucial for itolizumab and MEM98. Analysis of binding kinetics revealed that itolizumab has a lower affinity compared with other CD6 domain 1 mAbs. We compared potential agonistic (triggering) and antagonistic (blocking) properties of CD6 mAbs in assays where the mechanism of action was well defined. CD6 domain 1 and 3 mAbs were equally effective in triggering interleukin‐2 production by a cell line expressing a chimeric antigen receptor containing the extracellular region of CD6. CD6 domain 1 mAbs hindered binding of multivalent immobilized CD166 but were inferior compared with blocking by soluble CD166 or a CD6 domain 3 mAb. Characterization of CD6 mAbs provides an insight into how their functional effects in vivo may be interpreted and their therapeutic use optimized. 相似文献
18.
Li X Egorina E Bertelsen EL Dahlen H Hannestad K 《Scandinavian journal of immunology》2004,60(1-2):121-133
The initial novel observation of this study was that most B cells of male BXSB lupus mice bear surface IgG2a(b) of extrinsic origin. To define the surface antigen, we here examine three (NZBxBXSB)F1-derived IgG2a(b) monoclonal antibodies (mAbs) selected for binding to cell surfaces. Surprisingly, all three mAbs bound the nucleosome (nuc) particle, the fundamental unit of chromatin and an early target of autoimmunity in systemic lupus erythematosus. Their tentative dissociation constant (K(d)) for soluble nuc particles was approximately 7 x 10(-10) m. The mAbs bound more weakly to both H2A-H2B-DNA and H3-H4-DNA complexes, and in immunoblot they stained all four core histones. The mAbs detected a surface antigen on all cell lines examined, present on viable cells. When stripped of nuc, and in the presence of DNase I, their binding to cell lines improved. Heparin displaced the antigen from the cell surface. In vivo, the three mAbs stained B cells of several BALB/c mice clearly stronger than the isotype control; this differential staining was significantly reduced in FcgammaRIIB-deficient mice. The results indicate that the three mAbs recognize (a) planted antigen on viable cultured cells and (b) soluble autoantigen in vivo, leading to immune complexes that bind to FcgammaRIIB. Further experiments demonstrated that antinuc IgG2a could be eluted from splenocytes of a male BXSB lupus mouse. Hence, at least part of the extrinsic IgG2a(b) found on BXSB B cells may represent FcgammaRIIB-bound nuc-IgG2a(b) complexes. 相似文献
19.
B A Vance K H Karlson P M Morganelli P M Guyre 《Journal of immunological methods》1989,118(2):287-296
Interferon-gamma (IFN-gamma) activation of human monocytes in vitro results in enhanced phagocytosis and cellular cytotoxicity. These enhanced effector functions are attributable, at least in part, to increased expression of recognition molecules on the plasma membrane. In this article we report a rapid screening procedure for the primary selection of monoclonal antibodies (mAbs) which bind to cell surface molecules, the expression of which is increased or decreased by IFN-gamma. The procedure is based on flow cytometric analysis of mixed cell populations. Mouse mAbs were prepared using human monocytes cultured for 40 h with 400 U/ml of IFN-gamma as the immunogen. The hybridoma supernatants were screened using mixtures of six cell populations, some of which were pretreated with IFN-gamma for 40 h. Cells included in the mixture were chosen for their distinctive light scatter profiles. mAbs of interest were identified by preferential binding to monocytes and increased or decreased binding to monocytes treated with IFN-gamma. This procedure allowed us to screen several hundred clones per day, and to immediately eliminate mAbs that bound to B cells, T cells, neutrophils, and several cell lines. We selected ten mAbs which bound to surface molecules on monocytes that were modulated by IFN-gamma. Further characterization of five of the initial ten mAbs revealed that mAb gamma M phi 22.2 and mAb gamma M phi 197.1 bind to the high affinity Fc receptor for IgG (Fc gamma RI). mAb gamma M phi 28.3 appears to bind to a class II histocompatibility antigen and mAb gamma M phi 150.3 and mAb gamma M phi 195.18 appear to have binding patterns to human leukocytes and cell lines which are distinct from previously described mAbs. This rapid and specific procedure for screening mAbs has broad application for selecting mAbs that are specific for any given cell type and/or for surface molecules that are modulated by any cytokine and other hormone. 相似文献
20.
抗李斯特菌溶血素单克隆抗体的制备及初步鉴定 总被引:1,自引:0,他引:1
目的: 研制抗李斯特菌溶血素(LLO)的单克隆抗体(mAb).方法: 通过诱导重组大肠杆菌BL21(pGEX-6p-1-hly), 以SDS-PAGE分离菌体蛋白, 切割目的蛋白LLO-GST条带, 研磨粉碎后免疫BALB/c小鼠, 取免疫鼠脾细胞与Sp2/0细胞融合.利用亲和层析法纯化目的蛋白, 作为检测抗原进行杂交瘤细胞的筛选.采用间接ELISA法测定腹水效价, Dot-ELISA、 Western blot分析mAb的特异性.结果: 获得3株稳定分泌抗LLO mAb的杂交瘤细胞株3B6、 4D1、 5D10, 其Ig亚类均为IgG1.3B6、 4D1、 5D10腹水的ELISA效价分别为1:2×105、 1:2×105、 1∶1×105.Western blot试验中, 3株mAb均能与融合蛋白LLO-GST发生反应出现特异性条带, 而与纯化蛋白GST不反应.在Dot-ELISA试验中, 3株mAb均能与表达LLO的细菌发生特异性反应.结果表明, mAb 3B6、 4D1、 5D10是针对LLO的特异性mAb.结论: 成功地制备了抗LLO的mAb, 为进一步研究LLO的生物学特性、产单核细胞李斯特菌的致病机制, 以及建立产单核细胞李斯特菌(LM)快速检测技术奠定了基础. 相似文献