Generation of monoclonal antibodies against soluble human T cell receptor polypeptides.

One approach to the diagnosis and therapy of T cell-mediated diseases is to develop reagents specific for T cell receptor (TcR) variable (V) regions. To date, however, TcR expressed on the surface of antigen-specific T lymphocytes have proven to be poorly immunogenic. As a result, few monoclonal antibodies (mAb) recognizing human variable regions are available. In this report, we have used the "phosphatidylinositol linkage" strategy to generate soluble forms of two human allogeneic TcR derived from human cytotoxic T lymphocytes (CTL) known to be specific for HLA-A2 and HLA-Aw68/HLA-Aw69, respectively. Monomeric TcR alpha and beta chains from the HLA-A2-specific CTL were purified in large quantities from CHO cells and each was used to immunize mice to generate mAb. In particular, the anti-beta chain mAb, denoted anti-V beta 13, stain a significant (approximately 5%) fraction of human peripheral blood alpha/beta T lymphocytes, immunoprecipitate native anti-A2 TcR molecules, and activate T cells transfected with the relevant alpha and beta chain cDNA. Anti-alpha chain mAb were also obtained against a constant region determinant which can immunoprecipitate detergent-solubilized polypeptides. In general, we find that immunizations with soluble protein are far superior to those with cells bearing TcR chimeras or in combination with the purified protein.     

阻断型抗人IgE单克隆抗体的制备及其表位分析

目的 利用基因重组抗原免疫小鼠,制备抗人IgE单克隆抗体,研究其特异性和功能.方法 用表达的重组人IgE-Fc免疫BLAB/c小鼠,取其脾细胞与Sp2/0细胞融合,以间接ELISA法筛选杂交瘤上清.用ELISA阻断分析鉴定其生物学功能;通过丙氨酸突变扫描分析确定其抗原表位.结果 利用杂交瘤技术获得了1株抗人IgE的单克隆抗体178,它具有IgE抗体特异性反应,其识别的抗原表位位于IgE与其受体结合的关键部位.经鉴定其具有体外阻断IgE与其受体结合的活性.结论 利用基因重组抗原制备了1株抗人IgE的单克隆抗体178,此单克隆抗体具有体外阻断IgE与其受体结合的生物活性.     

人绒毛膜促性腺激素单克隆抗体的研制

目的获得针对人绒毛膜促性腺激素单克隆抗体。方法hCG蛋白免疫Balb／c小鼠,取其脾细胞与同系小鼠骨髓瘤细胞NS-1按8：1比例融合,间接ELISA法筛选阳性克隆,有限稀释法进行克隆化培养;制备腹水抗体;采用间接ELISA法鉴定抗体亚型和测定抗体效价。结果得到6株能稳定分泌单克隆抗体的杂交瘤细胞株;抗体经鉴定均为IgG1、κ型,效价均达10^-5以上。结论所获得的6株杂交瘤细胞株均有较强稳定分泌抗-hCG单克隆抗体的能力。这为有关hCG的检测、hCG本身相关研究以及避孕疫苗的研制打下了基础。     

抗人粘膜地址素-1单克隆抗体的制备与初步鉴定

目的制备抗人ＭＡｄＣＡＭ－１单克隆抗体（ｍＡｂ）。　方法以重组人ＭＡｄＣＡＭ－１胞外区蛋白免疫Ｂａｌｂ／ｃ小鼠，采用杂交瘤技术制备ｍＡｂ。结果获得１０株可分泌特异性ｍＡｂ的杂交瘤细胞，其腹水ｍＡｂ的效价为３．２×１０５～１．９×１０６，ｍＡｂ的Ｉｇ亚类均为ＩｇＧ１，其中，８株ｍＡｂ的轻链属κ型，２株为λ型。应用于免疫组化染色法，检测表明，正常成人小肠、附睾及前列腺组织中存在ＭＡｄＣＡＭ－１阳性表达的血管内皮细胞。结论　成功地研制出抗人ＭＡｄＣＡＭ－１的ｍＡｂ，为进一步研究ＭＡｄＣＡＭ－１在肠道黏膜免疫中的作用提供了有用的试剂。     

抗人DcR3单克隆抗体的研制及其鉴定

目的:制备抗人DcR3单克隆抗体(mAb),并鉴定其特异性.方法:纯化的His-DcR3融合蛋白免疫小鼠,应用淋巴细胞杂交瘤技术制备抗人DcR3 mAb并进行纯化,纯化后的抗体经Western blot和ELISA方法鉴定其特异性、Ig亚型和效价.结果:获得5株稳定分泌抗DcR3 mAb的杂交瘤细胞,均属IgG1亚型,其腹水抗体效价为1×10-5 ～1×10-7,Western blot显示5株细胞分泌的mAb均可识别DcR3蛋白,其中1株(1B1)可与SW480细胞成分反应.结论:成功建立稳定分泌抗人DcR3 mAb的杂交瘤细胞株,其分泌的抗体特异性强、效价高,为研究DcR3在组织中的表达、分布及研制ELISA试剂盒奠定基础.     

Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens

Infection with Helicobacter pylori is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of H. pylori gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against H. pylori cell lysate and the H. pylori urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an H. pylori-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the H. pylori Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the H. pylori urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related H. pylori binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following H. pylori infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of H. pylori antigens to be determined, which might be of use for vaccine development.     

重组人心肌肌钙蛋白I单克隆抗体的制备及鉴定

目的 制备重组人心肌肌钙蛋白I(rhcTnI)的单克隆抗体,并对其单克隆抗体的特性进行鉴定.方法 利用纯化后的rhcTnI作为免疫原,常规免疫Balb/c小鼠,取其脾细胞与同系骨髓瘤细胞Sp2/0进行常规融合,用甲基纤维素半固体培养基法获得单克隆抗体杂交瘤细胞株,利用ELISA、Western blot等方法对单克隆抗体的特性进行鉴定.结果 筛选出3株稳定分泌抗rhcTnI的单克隆抗体杂交瘤细胞株,分别命名为4B3、7D5和3E6.3株单克隆抗体的免疫球蛋白亚类分别为IgG1(4B3、7D5)和IgG2b(3E6).3株单克隆抗体与rhcTnI发生特异性结合,而与大肠杆菌菌体蛋白、乳酸脱氢酶(LDH)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)无特异性交叉反应,3株单克隆抗体的亲和力常数分别为1.76×109Umol、143×109L/mol和1.04×109L/mol.结论 获得了效价高、特异性好的抗rhcTn I的单克隆抗体,为cTnI诊断试剂盒的研制奠定了基础.     

重组人心肌肌钙蛋白Ⅰ单克隆抗体的制备及鉴定

目的制备重组人心肌肌钙蛋白I（rhcTnI）的单克隆抗体,并对其单克隆抗体的特性进行鉴定。方法利用纯化后的rhcTn I作为免疫原,常规免疫Balb／c小鼠．取其脾细胞与同系骨髓瘤细胞Sp2／0进行常规融合。用甲基纤维素半固体培养基法获得单克隆抗体杂交瘤细胞株,利用ELISA、Western blot等方法对单克隆抗体的特性进行鉴定。结果筛选出3株稳定分泌抗rhcTnI的单克隆抗体杂交瘤细胞株,分别命名为4B3、7D5和3E6。 3株单克隆抗体的免疫球蛋白亚类分别为IgGl（4B3、7D5）和IgG2b（3E6）。3株单克隆抗体与rhcTnI发生特异性结合,而与大肠杆菌菌体蛋白、乳酸脱氢酶（LDH）、肌酸激酶（CK）、肌酸激酶同工酶（CK-MB）无特异性交叉反应,3株单克隆抗体的亲和力常数分别为1．76×10^9L／mol、1．43×10^9L/mol和1．04×10^9L/mol。结论获得了效价高、特异性好的抗rhcTnI的单克隆抗体,为cTnI诊断试剂盒的研制奠定了基础。     

C-myc单克隆抗体的制备及鉴定

目的制备与鉴定鼠抗C-myc蛋白单克隆抗体。方法利用C-myc蛋白做免疫原免疫Balb/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,经间接ELISA法筛选、有限稀释法克隆、单克隆抗体Ig亚型鉴定,获得2株能够稳定分泌抗C-myc特异性抗体的杂交瘤细胞株,再将杂交瘤细胞注射入小鼠腹腔诱使小鼠产生腹水。结果获得2株可稳定分泌抗C-myc抗体的杂交瘤细胞株,命名为SHH-1H11,SHH-2A9,Ig亚型分别为IgG1亚类和IgM。细胞培养上清效价分别为1:10240、1:5 120;小鼠腹水效价分别为1:64 000、1:32 000。结论制备的C-myc单克隆抗体仅与C-myc蛋白反应,与其他蛋白没有交叉反应,表明获得的单克隆抗体的特异性很高。     

雪卡毒素单克隆抗体的制备及鉴定

目的制备雪卡毒素的单克隆抗体并对其生物学特性进行鉴定。方法以雪卡毒素类似物莫能菌素为半抗原,分别采用混合酸酐法将其与载体牛血清白蛋白(BSA)偶联合成免疫抗原M-BSA,碳二亚胺法与卵清白蛋白(OVA)偶联合成包被抗原M-OVA。以M-BSA免疫Balb/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合制备杂交瘤。以自制雪卡毒素提取物为竞争抗原,间接竞争ELISA法筛选稳定分泌雪卡毒素抗体的特异性细胞株。小鼠体内诱生腹水,辛酸硫酸铵沉淀法进行纯化。采用抗体亚型鉴定试剂盒鉴定所得抗体的亚型,间接竞争ELISA法鉴定抗体的特异性和交叉性。结果获得3株稳定分泌抗雪卡毒素抗体的杂交瘤细胞株1D5、2G11、3G11,其Ig亚型均属于IgG1亚型。交叉反应结果表明3株单克隆细胞产生的抗体除与莫能菌素有较高的交叉反应外,与其他雪卡毒素类似物均无明显交叉反应。结论成功筛选到了分泌雪卡毒素抗体的细胞株,为雪卡毒素免疫分析方法的进一步研究奠定基础。     

Generation of human monoclonal antibodies that confer protection against pertussis toxin.

A panel of human monoclonal antibodies reactive with pertussis toxin has been generated by means of Epstein-Barr virus infection. One of these, the 3F11 monoclonal antibody, showed the ability to neutralize in vitro and in vivo the toxic effects of the toxin. Western blot (immunoblot) analysis located the 3F11 epitope on the S3 subunit.     

Generation of monoclonal antibodies against a human T cell receptor beta chain expressed in transgenic mice.

The generation of a panel of monoclonal antibodies specific for different variable (V) regions of human T cell receptors will be of great importance in the study of T cell-mediated diseases. However, relatively few such reagents exist, due in part to the poor immunogenicity of TcRs on the surface of human T cells. We have employed a strategy in which T cells from a transgenic mouse line expressing a human V beta 3 C beta 1 TcR were used to immunise syngeneic conventional mice to generate two monoclonal antibodies specific for human T cell receptors. Binding of antibody JOVI.3, which stained approximately 5% of human peripheral blood CD3 positive T cells, correlated with the expression of the human TcR V beta 3 gene segment. Antibody JOVI.1 recognised a determinant on the majority of TcRs, staining 50-75% of peripheral blood T cells and T cell lines expressing different V beta regions. Some TcRs, however, failed to react with this antibody. Both antibodies immunoprecipitated detergent-solubilised TcR molecules and were capable of inducing proliferation of peripheral blood T cells.     

抗RAET1G2单克隆抗体的研制和初步鉴定

目的:制备鼠源抗RAET1G2单克隆抗体,并对其特异性进行鉴定。方法:以原核表达的RAET1G2蛋白为抗原免疫BALB/c小鼠,运用B淋巴细胞杂交瘤技术制备抗RAET1G2单克隆抗体;用免疫双扩散方法鉴定Ig亚类;Western blot鉴定单克隆抗体的特异性。结果:获得了4株可分泌特异性抗RAET1G2抗体的杂交瘤细胞株7A11、9C6、10F9和12F8,Ig亚类均为IgG1。结论:制备的杂交瘤细胞株能稳定分泌特异性的抗RAET1G2抗体,并且所分泌的单克隆抗体都能识别天然的RAET1G蛋白,为进一步研究游离性的RAETlG2分子与肿瘤进展的关系以及分析各种肿瘤细胞表面的RAET1G表达情况创造了条件。     

抗鸡蛋卵类黏蛋白单克隆抗体的制备与鉴定

目的制备和鉴定鼠抗鸡蛋主要过敏原卵类黏蛋白的单克隆抗体。方法利用鸡蛋卵类黏蛋白(ovm)为免疫原,免疫Balb/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞。用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA、Western blotting鉴定该单克隆抗体的特性和交叉性。利用双抗体夹心ELISA法检测食品中的鸡蛋过敏原。结果获得4株可稳定分泌鼠抗卵类黏蛋白的单克隆抗体,分别命名为1G8,2H5,3A7,4G4,其Ig亚型均为IgG1。且4株单抗效价均在10-8以上。ELISA和Western blotting分析表明该4株单抗均能特异性识别卵类黏蛋白,并且建立双抗体夹心ELISA的方法可以准确的检测出食品中鸡蛋过敏原的存在。结论成功制备了4株高效价的鼠抗卵类黏蛋白的单克隆抗体,为建立卵类黏蛋白检测及纯化方法奠定了基础,也可为食品中鸡蛋过敏原的检出提供依据。     

抗急性出血性结膜炎病毒受体单克隆抗体的制备与鉴定

用细胞保护试验、间接免疫荧光试验及流式细胞仪等方法，对采用细胞融合技术所得到的抗肠道病毒７０型（Ｅｖ７０）和柯萨奇病毒Ａ２４型变异株（ＣＡ２４ｖ）受体的单克隆抗体进行了研究，发现４株抗Ｈｅｌａ细胞膜受体的单克隆抗体能同时阻断Ｅｖ７０和ＣＡ２４ｖ某些毒株感染ＨｅＬａ细胞。这一结果提示ＥＶ７０和ＣＡ２４存在有相似的受体，对这两种不同的病毒能引起同一种临床疾病的发病机制的阐明有一定的指导意义。     

Generation of genetic engineering monoclonal antibodies against prion protein

Two strains of Fab monoclonal antibodies (mAbs) against prion protein, designated as IV-66 and IV-78, were selected from the phage display libraries. The gene sequences encoding the light κ chain and heavy Fd chain of IV-78 were inserted into a baculovirus expression cassette vector for mouse IgG expression. Western blot, Dot-ELISA and immunoprecipitation confirmed that these Fab and IgG mAbs reacted well with the recombinant hamster and human PrP proteins expressed in prokaryotic and in mammalian cells and PrP^Sc from scrapie-infected hamsters. It demonstrates that mAbs against prion protein are successfully generated by phage-display technique.     

Generation and characterization of monoclonal antibodies against Staphylococcus aureus thermonuclease

Monoclonal antibodies (MAbs) against Staphylococcus aureus thermonuclease (TN) were raised by immunizing BALB/c mice with a commercial TN preparation. Six monoclones were generated producing MAbs specific for S. aureus TN as tested in Western blots and ELISA. They all combined with a 17 kD and a 21 kD protein, respectively, both of which showed DNase activity. All MAbs were of IgG1 isotype with kappa light chain. Competition ELISA showed that five of the MAbs recognized a total of three different binding sites of TN, designated I, II and III, respectively. Only the anti-site II MAbs inhibited the DNase activity. A MAb-based sandwich ELISA showed a lower detection limit for TN of approximately 0.5 ng/ml protein. Only S. aureus strains (culture supernatants) showed positive ELISA (31 positive/31 tested), although other tested gram positive cocci produced thermostable nucleases. The MAbs have potentials as reagents for rapid and specific detection of S. aureus.     

抗人乳腺癌细胞膜表面角蛋白CK8的单克隆抗体的制备及鉴定

目的：建立针对多药耐药人乳腺癌细胞膜表面差异抗原的抗体库,并鉴定抗角蛋白CK8特异性的单克隆抗体。方法：采用细胞免疫和杂交瘤技术制备单克隆抗体库,经免疫沉淀得到抗原-抗体复合物,SDS-PAGE分离目的抗原,质谱测序,Western blot验证及激光共聚焦显微镜观察抗原分子的细胞膜定位。结果：成功筛选到稳定分泌抗CK8分子的单克隆抗体的杂交瘤细胞株（7F9）。结论：文中所研制的单克隆抗体具有与乳腺癌细胞膜表面CK8分子特异结合的活性,在上皮来源肿瘤标志物CK8的临床检测及CK8相关生物学活性研究中具有广泛应用前景。     

抗人补体膜攻击复合物新抗原单克隆抗体的制备及鉴定

目的　研制针对人补体膜攻击复合物 (MAC)新抗原的特异性单克隆抗体。方法　在兔红细胞表面组装人MAC ,分离纯化RBC膜蛋白。以初步纯化的MAC免疫Balb c小鼠 ,取免疫小鼠脾细胞与SP2 0骨髓瘤细胞融合 ,采用免疫斑点杂交的方法分别以纯化的单一补体成分C5、C6、C7、C8、C9和MAC同时进行阴性和阳性筛选。结果　获得了 2株仅与MAC反应而不与各单体成分反应的单克隆抗体。进一步以体外组装的补体终末复合物进行鉴定 ,证实所获单克隆抗体可特异性识别MAC复合物中C9分子暴露出的新抗原。经ELISA法测定 ,2株杂交瘤细胞的培养上清液和腹水效价分别为 1× 1 0 -3 ,1× 1 0 -3和 1× 1 0 -6,1× 1 0 -7;单克隆抗体的重链均属小鼠IgG1亚类 ,轻链均为κ型。结论　获得了 2株特异性识别MAC新抗原的单克隆抗体。     

Characterization of monoclonal antibodies against human lactoferrin

The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties.