登革2型病毒单克隆抗体的制备与鉴定

用登革2型病毒参考株免疫的BALB/C小鼠脾细胞与SP2/0小鼠骨髓瘤细胞在PEG1000作用下进行融合,获得了10株分泌抗登革2型病毒单克隆抗体的杂交瘤。经间接免疫荧光鉴定,有4株杂交瘤产生的单克隆抗体对登革2型病毒是型特异的;2株对登革1型有交叉反应;1株对登革3型有交叉反应;1株对登革4型有交叉反应;另2株为黄病毒属特异的。经补体结合和血凝抑制试验鉴定,有2个单克隆抗体是黄病毒属血凝抑制特异的。这10个单克隆抗体对登革热的诊断是很有用的。     

A perspective of monoclonal antibodies: past, present, and future

In 1975, the development of the technique to produce monoclonal antibodies revolutionized the approach to cancer detection and therapy. Hundreds of monoclonal antibodies to the epitopes of tumor cells have been produced, providing more specific tools for probing the cellular elements of cancer. At the same time, these tools have disclosed greater complexity in the character of these cells and stimulated further investigation. Although there are antibodies to specific epitopes of neoplastic cells, this purity has not provided the improved detection and therapy of cancer first expected. Technical manipulations have provided limited improvement in results, but more sophisticated techniques, such as biologic response modifiers, may be required to attain clinical results that can be universally applied. The intense research in monoclonal antibodies and their application does offer promise that the goal of improved cancer detection and therapy will be forthcoming.     

登革1型病毒的单克隆抗体

我们用SP2/0-Ag14小鼠骨髓瘤细胞与用登革1型病毒免疫的BALB/C小鼠脾细胞在融合剂PEG-1000的诱导下进行融合,获得了15株产生抗登革1型病毒单克隆抗体的杂交瘤细胞系。其中14株属登革病毒型特异,1株属登革病毒亚群特异,它们的荧光效价均在10,240以上。15株杂交瘤均无血凝活性,3株有补体结合活性。1D_4,1B_9的IgG亚类为IgG_1,1F_(11)为IgG_2a,轻链均为K型。1株杂交瘤(1D_4)的染色体数为87～115。用SDS-PAGE测定1D_4分子量,重链为54,000,轻链为24,000。15株杂交瘤连续传代3～6个月,冻存复苏后抗体水平未见下降。     

新型抗uPAR人源化抗体的表达和活性检测

目的制备抗尿激酶型纤溶酶原激活物受体（uPAR）人源化抗体并初步检测它们与抗原的亲和能力。方法通过计算机辅助设计的结果,合成新型抗uPAR抗体的轻链和重链可变区基因序列,通过重叠PCR方法,拼接成完整的轻链和重链基因并克隆入pIRES双向表达载体。瞬时转染293T细胞,收取细胞上清,rProtein A亲和层析法纯化目的抗体,并进行SDS-PAGE和免疫印迹鉴定,采用Biacore3000技术检测抗体与抗原的结合能力。结果成功构建5种表达载体S1~S5,纯化的抗体在还原SDS-PAGE中表现为相对分子质量约为25×103和55×103两条带;免疫印迹分析表明,该人源化抗体可与羊抗人IgG特异性结合。Biacore3000实验结果表明,S2、S4和S5抗体与抗原具有良好的亲和活性,且亲和活性分别为1.74×10-8,1.49×10-8和1.05×10-8mol/L。结论成功构建并表达了5种抗uPAR人源化抗体,其中S2、S4和S5具有良好的抗原结合能力。     

Bexxar治疗非霍奇金淋巴瘤的研究现状

B细胞靶向特异性单克隆抗体的出现为惰性淋巴瘤的治疗提供了新的策略。由于淋巴瘤对辐射较为敏感,在过去的10年中,应用131I标记抗-CD20单克隆抗体(商品名:Bexxar)对化疗无效的非霍奇金淋巴瘤(NHL)患者进行放射免疫治疗取得了长足的发展。     

抗工程菌产HBcAg单克隆抗体杂交瘤细胞系的建立

本文用纯化的工程菌产HBcAg免疫Balb/C小鼠,将该鼠的脾淋巴细胞与鼠骨髓瘤细胞在聚乙二醇作用下融合,获得2株抗-HBc阳性、与工程菌茵体蛋白无交叉反应的杂交瘤细胞系.2个亚株经体外培养两月余,传代20次,液氮反复冻存、复苏3次,仍能稳定分泌抗-HBc.该细胞接种经石蜡油致敏的Balb/C小鼠腹腔,产生抗-HBc腹水.其中1株经鉴定,其抗体亚类为IgG2a.     

抗莱氏无胆甾原体单克隆抗体杂交瘤细胞株的建立

本文采用浓缩提纯的莱氏无胆甾原体做为抗原,多途径免疫 BALB/C 小鼠,取脾细胞与 SP_2/O-Ag~(14)小鼠骨髓瘤细胞融合,建立了4株分泌抗莱氏无胆甾原体单克隆抗体杂交瘤细胞。分析了杂交瘤细胞的染色体,平均数为103条;制备了腹水抗体,经 APAAP 法(碱性磷酸酶—抗碱性磷酸酶桥联酶标显色技术)和 IFA 法检测,抗体效价在80～320之间;对免疫球蛋白亚类进行了鉴定,其抗体均为 IgM。上述细胞经3～4个月稳定传代后,冻存于液氮中。该单克隆抗体的制备对细胞培养中支原体污染的检出可能有重要意义。     

人附睾蛋白4单克隆抗体与免放试剂盒的制备及其对卵巢癌诊断的临床价值探讨

目的 制备人附睾蛋白4(HE4)单克隆抗体并研制免放分析试剂盒, 探讨其在卵巢癌临床诊断中的价值。 方法 用重组人HE4免疫小鼠, 通过细胞融合杂交瘤技术制备并筛选可以配对的HE4单抗, 用125I标记其中一株单抗, 研制HE4免放试剂盒, 并评价其检测的灵敏度。通过检测临床血清标本来评价其对卵巢癌诊断的灵敏度和特异度。 结果 筛选出了一对亲和力与特异性高的HE4单抗, 其免放试剂盒检测的灵敏度为3.65 pmol/L, 其对卵巢癌的临床诊断的灵敏度可达90.83%, 特异度为99.17%。 结论 制备的单克隆抗体亲和力高, 研制的免放试剂盒各项指标较好, 可用于卵巢癌的早期临床诊断和疗效观察, 降低卵巢癌患者的病死率。     

抗Salmonella minnesota R_(595)菌脂多糖杂交瘤的建立及其抗体特性的初步鉴定

将煮沸致死的R_(595)菌体免疫BALB/C小鼠,取其脾细胞在PEG介导下与SP_(2/0)细胞融合,经筛选获得2个阳性孔2D_6、3H_4。将2D_6、3H_4阳性孔细胞克隆,传代最终获得8株能稳定分泌抗R_(595)内毒素单抗的杂交瘤株。经鉴定,杂交瘤细胞染色体数90余条,抗体效价(EL1SA法)10~4～10~5,2D_6B_1,2D_6E_7单抗的Ig类型为IgG_1,3H_42F_7、3H_42H_3单抗Ig类型为IgG2_b,轻链均为K链。类脂A抑制试验显示,单抗识别的表位为类脂A或KDO,表明抗体识别内毒素保守区域。间接EL1SA试验表明,抗体能与多种GNB交叉反应,提示抗体具广谱交叉反应性。玻片凝集试验显示,抗体仅能凝集R_(595)菌,不能凝集其它GNB菌。     

Antibody-based cancer treatment with ultra-short range Auger electron-emitting radionuclides: dual receptor and DNA targeting strategies

The long-heralded potential of targeted cancer treatment using monoclonal antibodies is finally being realized. Several antibodies are already used in the oncology clinic and many others are undergoing preclinical evaluation. In addition to the development of unconjugated antibodies, there is intense interest in the potential clinical use of antibodies as vehicles for targeting cytotoxic agents specifically to cancer cells. For example, radioimmunotherapy which involves the use of antibodies to deliver radionuclides to target cells is an approved treatment modality for cancer. Our laboratory is involved in developing technologies for radioimmunotherapy using a unique class of radionuclides, known as Auger electron emitters. A key feature of the Auger electrons emitted by these radionuclides is that they traverse very small ranges (molecular dimensions) in biological tissues. The emission of Auger electrons results in a gradient of energy deposition with the majority of the radiochemical damage occurring in the immediate vicinity (within a few cubic nanometers) of the decaying radionuclide. Therefore, realizing the full potential of Auger electron emitting isotopes in radioimmunotherapy requires more sophisticated approaches than directly radiolabeling anticancer antibodies. Strategies which involve targeting the radionuclide not only to cancer cells but also to the DNA of those cells are necessary. In this paper potential dual, receptor and DNA, targeting systems for radioimmunotherapy with Auger electron-emitting radionuclides are discussed.     

Immunoreactivity assay for labeled anti-melanoma monoclonal antibodies

A convenient, rapid, and reproducible assay was developed to evaluate the immunoreactivity of radiolabeled monoclonal antibodies against three different human melanoma-associated antigens, p97, a proteoglycan and a GD3 ganglioside. A cloned melanoma cell line (M 2669 CL 13) was selected as the target and, when fixed with paraformaldehyde, showed binding as good as or better than that obtained with live cells for the three antigens. Fixed cells retained good binding properties stored at 4 degrees C for over 6 mo. This assay has general applicability to other antigen-antibody systems for testing chemically modified monoclonal antibodies or fragments during the development of a radiopharmaceutical or as a routine quality control measure for clinical agents.     

脾内注射法免疫小鼠制备抗—HBc单克隆抗体的研究

用基因工程菌产乙型肝炎核心抗原(HBcAg)对 Balb/c 小鼠脾内多点注射法免疫,将该鼠的脾淋巴细胞与鼠骨髓瘤细胞在 PEG 作用下融合,用固相放射免疫分析法(SPRIA)筛选,成功地获得了2株抗-HBc 阳性的杂交瘤细胞株。通过中和试验和交叉鉴定,2株抗-HBc 单克隆抗体只能与 HBcAg 起反应,而不能与 HBsAg 和 HBeAg 反应。经多次亚克隆后,所得腹水中的抗-HBc 滴度达1:64000～1:28000。这2株单克隆抗体做包被抗体检测 HBcAg 和(125)~Ⅰ标记查患者血清标本时,所测得的结果与人抗-HBc 多克隆抗体做包被或(125)~Ⅰ标记所测得的结果相一致。2株杂交瘤细胞连续传代3个多月,冻存复苏后仍能稳定地分泌特异的抗-HBc 单克隆抗体。     

Inhibition of autoradiolysis of radiolabeled monoclonal antibodies by cryopreservation

Autoradiolysis of therapeutic doses of monoclonal antibodies can occur rapidly, limits their shelf life and makes onsite radiolabeling a near-necessity. We evaluated freezing of three different 131I-labeled murine monoclonal antibodies at -70 degrees C, immediately following radiolabeling, as a method of diminishing autoradiolysis, and of preserving immunoreactivity. Freezing greatly limits the ability of radiation-induced free radicals to diffuse in solution and thus produce radiolytic damage. By freezing at -70 degrees C autoradiolytic damage of immunoreactivity of three different 131I monoclonal antibodies could be largely eliminated, in contrast to the 80-90% losses in immunoreactivity seen with storage at 4 degrees C for a period of 1 to 12 days. Reduced in vitro deiodination rates are also seen for frozen antibodies. Limited studies with 125I-labeled antibodies indicate autoradiolysis does occur, though at a slower rate per mCi than for 131I, and that this process is also retarded by freezing. Freezing may be valuable while quality control procedures are performed following radiolabeling as well as if temporary storage or shipment of radioantibodies prior to patient dosing is undertaken. While the approach should be validated for each antibody studied, freezing of therapeutic doses of monoclonal antibodies appears to be a simple and effective approach to the problem of autoradiolysis.     

Future role of radiolabeled monoclonal antibodies in oncological diagnosis and therapy

This review discusses the current limitations and future prospects of radiolabeled antibodies in cancer imaging (radioimmunodetection, or RAID) and therapy (radioimmunotherapy, or RAIT). Aspects such as the antibody vehicle, antigen target, radiolabel, tumor, host, and RAID and RAIT procedures are considered. In the short timespan for the development of RAID, tumors as small as 0.5 cm, which are sometimes missed by other radiological methods, can now be imaged with antibody fragments labeled with suitable radionuclides (eg, 111In, 123I, and 99mTc), particularly when single photon emmission computed tomography (SPECT) scanning methods are employed. 99mTc is clearly the preferred label, and the recent development of simple and rapid methods to attach this isotope to antibodies should be a welcome advance for the more widespread use of RAID. In RAIT, radiosensitive neoplasms, such as lymphomas, are already showing impressive responses to 131I-labeled antilymphoma murine monoclonal antibodies. Therefore, the successful conjugation of beta- and alpha-emitters to "humanized" monoclonal antibodies should provide a new generation of promising cancer therapeutics.     

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with ^99mTc or ¹¹¹In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform ‘evidence-based biological therapy’ of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and follow-up.     

Monoclonal antibodies: old and new trends in breast cancer imaging and therapeutic approach

Over the last two decades, various research protocols were applied for scintigraphic imaging, prognosis and treatment of breast cancer, using monoclonal antibodies. Monoclonal antibodies approved by the United States Food and Drug Administration (FDA) include the anti-carcinoembryonic antigen (CEA), and B72.3, prepared against the tumour-associated glycoprotein, TAG-72. The recombinant humanized "cold" anti-HER2 monoclonal antibody (trastuzumab), which targets oncogene receptor HER2 has hitherto been the only monoclonal antibody widely used for the treatment of breast cancer in the USA, with or without chemotherapy. Trastuzumab is constructed against the HER2 oncogene receptor (also known as neu or c-erb-B2), which is overexpressed in 25%-30% of breast cancer cell lines and is associated with poor prognosis. Immuno-lymphoscintigraphy is also applied to guide and monitor the effect of treatment regimes. Radiolabelled, "hot" monoclonal antibodies are currently being applied for the treatment of primary or metastatic breast cancer, in experimental, pre-clinical, or clinical trials, in combination with traditional external beam radiotherapy and/or chemotherapy. Radioimmunotherapy comprises systemically administered monoclonal antibodies, linked to high-energy, beta-emitting radionuclides. Radioactive antibodies, in the form of yttrium-90 (90Y)-BrE-3, 90Y- m170 and 131I- or 90Y- labelled L6 antibody, are applied with adjuvant autologous peripheral blood stem cells transfusion, to prevent myelotoxicity. Partial or rarely complete responses to "hot" antibody treatment, of breast cancer have been reported. Innovative strategies using this combined-modality treatment hold promise for better disease-free and survival rates.     

A passive haemagglutination test for human anti-mouse antibody (HAMA) responses in patients undergoing immunoscintigraphy

Sera from seven patients with ovarian or colorectal carcinoma who had been given only single injections of 131I or 111In labelled IgG1, IgG2a or IgG2b monoclonal antibodies for immunoscintigraphy 2 weeks to 9 months earlier were tested for their ability to agglutinate sheep red blood cells coated with a mouse monoclonal antibody. Six showed agglutination, and this was still seen when sera were diluted to 1/125 in four patients and as low as 1/3125 in the other two. Sera from rats and mice which had been injected with monoclonal antibody also caused agglutination, which in the case of the mouse sera is due to the presence of anti-idiotypic antibody. This study shows that it is feasible to detect HAMA by a rapid, simple, passive haemagglutination reaction.     

Preadsorption of radiolabeled monoclonal antibodies to liver and spleen tissues leads to higher tumor-to-normal-tissue ratios

This study addresses the impact of background activity on the use of radioimmunoconjugates for radioimmunodiagnosis and radioimmunotherapy. Since the liver and the spleen represent organs with preferential nonspecific uptake, we exposed radiolabeled (iodinated and Indium-111 labeled) preparations of monoclonal antibodies to a suspension of fresh liver and spleen cells at physiological temperature and compared their immunoreactivity, in vivo biodistribution, and tumor targeting to those of the same radiolabeled proteins without prior adsorption to this suspension. The biodistribution studies were performed under conditions of high background activity, i.e., shortly after the injection (1 hour) and using a high dose of the protein. Preadsorption of radiolabeled monoclonal antibodies results in a significant decreased uptake in certain normal tissues, i.e., greater contrast between normal and tumor tissues, as demonstrated by the quotient of the two target-to-nontarget ratios (exposed/unexposed antibody) which was greater than one for most of the tissues examined.     

抗内甲基转移酶单克隆抗体的制备及临床应用研究

目的：建立抗人甲基转移酶单克隆抗体细胞株并建立相应的蛋白测定方法,观察脑瘤组织中O^6-甲基鸟嘌呤－DNA甲基转移酶（MGMT）表达水平与亚硝脲药物疗效的关系,方法：采用细胞融合技术建立抗人甲基转移酶单克隆抗体细胞胞株,采用免疫组织化染色方法,检测60例脑瘤组织中MGMT表达水平,结果：得到了7株分泌抗人甲基转移酶单克隆抗体的杂交瘤细胞株,并建立了测定MGMT免疫组化方法,观察的60例脑瘤组织中有27例MGMT阴性,经亚硝脲药物治疗,其中24例好转,3例复发,另3例MGMT可疑阳性的患者,经亚硝脲药物治疗,其中2例好转,1例复发,25例MGMT阳性患者,经亚硝脲药物治疗,5例好转,12例复发,8例死亡,5例MGMT为强阳性患者,使用亚硝脲药物治疗无效,全部死亡,结论：建立了7株稳定分泌抗人甲基转移酶单克隆抗体的杂交瘤细胞株及MGMT蛋白测定方法,为临床开展亚硝脲预见性化疗提供了必要手段,初步结果显示,脑瘤组织中MGTM表达水平与亚硝脲药物疗效及预后有关,是肿瘤细胞对亚硝脲药物产生耐药性的基础。     

Radioimmunotherapy of B-cell non-Hodgkin's lymphoma.

In the past decade, several new antibody-based therapies - using either radiolabelled or unlabelled monoclonal antibodies - have become available for the treatment of patients with refractory or recurrent non-Hodgkin's lymphoma (NHL). Unlabelled monoclonal antibodies (mAbs) kill lymphoma cells by activating host immune effector mechanisms, or by inducing apoptosis. These mAbs can also be used to guide radionuclides to the lymphoma. This radioimmunotherapy (RIT) has been studied with various nuclides (131I, 90Y, 67Cu and 186Re) and with various mAbs. In this review the radionuclides, methods of dosing and recent RIT studies in patients with B-cell NHL are reviewed. Most of these studies demonstrate that RIT is an effective new treatment modality for NHL.